Modulators of PNPLA3 expression

ABSTRACT

The present embodiments provide methods, compounds, and compositions useful for inhibiting PNPLA3 expression, which may be useful for treating, preventing, or ameliorating a disease associated with PNPLA3.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0317USLSEQ_ST25.txt created Sep. 13, 2018, which is 480 kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD

The present embodiments provide methods, compounds, and compositions useful for inhibiting PNPLA3 (patatin like phospholipase domain containing 3; hypothetical protein dJ796I17.1; adiponutrin; DJ796I17.1) expression, and in certain instances, reducing the amount of PNPLA3 protein in a cell or animal, which can be useful for treating, preventing, or ameliorating a disease associated with PNPLA3.

BACKGROUND

Non-alcoholic fatty liver disease (NAFLD) covers a spectrum of liver disease from steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. NAFLD is defined as fat accumulation in the liver exceeding 5% by weight, in the absence of significant alcohol consumption, steatogenic medication, or hereditary disorders (Kotronen et al, Arterioscler Thromb. Vasc. Biol. 2008, 28: 27-38).

Non-alcoholic steatohepatitis (NASH) is NAFLD with signs of inflammation and hepatic injury. NASH is defined histologically by macrovesicular steatosis, hepatocellular ballooning, and lobular inflammatory infiltrates (Sanyal, Hepatol. Res. 2011. 41: 670-4). NASH is estimated to affect 2-3% of the general population. In the presence of other pathologies, such as obesity or diabetes, the estimated prevalence increases to 7% and 62% respectively (Hashimoto et al, J. Gastroenterol. 2011. 46(1): 63-69).

PNPLA3 is a 481 amino acid member of the patatin-like phospholipase domain-containing family that is expressed in the ER and on lipid droplets. In humans, PNPLA3 is highly expressed in the liver, whereas adipose tissue expression is five-fold less (Huang et al, Proc. Natl. Acad. Sci. USA 2010. 107: 7892-7).

SUMMARY

Certain embodiments provided herein are compounds and methods for reducing the amount or activity of PNPLA3 mRNA, and in certain embodiments, reducing the amount of PNPLA3 protein in a cell or animal. In certain embodiments, the animal has a liver disease. In certain embodiments, the disease is NASH. In certain embodiments, the disease is NAFLD. In certain embodiments, the disease is hepatic steatosis. In certain embodiments, the disease is liver cirrhosis. In certain embodiments, the disease is hepatocellular carcinoma. In certain embodiments, the disease is alcoholic liver disease. In certain embodiments, the disease is alcoholic steatohepatitis (ASH). In certain embodiments, the disease is HCV hepatitis. In certain embodiments, the disease is chronic hepatitis. In certain embodiments, the disease is hereditary hemochromatosis. In certain embodiments, the disease is primary sclerosing cholangitis. Certain compounds provided herein are directed to compounds and compositions that reduce liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation in an animal.

Certain embodiments provided herein are directed to potent and tolerable compounds and compositions useful for inhibiting PNPLA3 expression, which can be useful for treating, preventing, ameliorating, or slowing progression of liver diseases. Certain embodiments provided herein are directed to compounds and compositions that are more potent or have greater therapeutic value than compounds publicly disclosed.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the embodiments, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and GenBank and NCBI reference sequence records are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

It is understood that the sequence set forth in each SEQ ID NO in the examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Compounds described by ION number indicate a combination of nucleobase sequence, chemical modification, and motif.

Definitions

Unless otherwise indicated, the following terms have the following meanings:

“2′-deoxynucleoside” means a nucleoside comprising 2′-H(H) furanosyl sugar moiety, as found in naturally occurring deoxyribonucleic acids (DNA). In certain embodiments, a 2′-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil).

“2′-O-methoxyethyl” (also 2′-MOE) refers to a 2′-O(CH₂)₂—OCH₃) in the place of the 2′-OH group of a ribosyl ring. A 2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-MOE nucleoside” (also 2′-O-methoxyethyl nucleoside) means a nucleoside comprising a 2′-MOE modified sugar moiety.

“2′-substituted nucleoside” or “2-modified nucleoside” means a nucleoside comprising a 2′-substituted or 2′-modified sugar moiety. As used herein, “2′-substituted” or “2-modified” in reference to a sugar moiety means a sugar moiety comprising at least one 2′-substituent group other than H or OH.

“3′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 3′-most nucleotide of a particular compound.

“5′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 5′-most nucleotide of a particular compound.

“5-methylcytosine” means a cytosine with a methyl group attached to the 5 position.

“About” means within +10% of a value. For example, if it is stated, “the compounds affected about 70% inhibition of PNPLA3”, it is implied that PNPLA3 levels are inhibited within a range of 60% and 80%.

“Administration” or “administering” refers to routes of introducing a compound or composition provided herein to an individual to perform its intended function. An example of a route of administration that can be used includes, but is not limited to parenteral administration, such as subcutaneous, intravenous, or intramuscular injection or infusion.

“Administered concomitantly” or “co-administration” means administration of two or more compounds in any manner in which the pharmacological effects of both are manifest in the patient. Concomitant administration does not require that both compounds be administered in a single pharmaceutical composition, in the same dosage form, by the same route of administration, or at the same time. The effects of both compounds need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive. Concomitant administration or co-administration encompasses administration in parallel or sequentially.

“Amelioration” refers to an improvement or lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. In certain embodiments, amelioration includes a delay or slowing in the progression or severity of one or more indicators of a condition or disease. The progression or severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.

“Animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

“Antisense activity” means any detectable and/or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound to the target.

“Antisense compound” means a compound comprising an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group. Examples of antisense compounds include single-stranded and double-stranded compounds, such as, oligonucleotides, ribozymes, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.

“Antisense inhibition” means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.

“Antisense mechanisms” are all those mechanisms involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.

“Antisense oligonucleotide” means an oligonucleotide having a nucleobase sequence that is complementary to a target nucleic acid or region or segment thereof. In certain embodiments, an antisense oligonucleotide is specifically hybridizable to a target nucleic acid or region or segment thereof.

“Bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety. “Bicyclic sugar” or “bicyclic sugar moiety” means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, the bicyclic sugar moiety does not comprise a furanosyl moiety.

“Branching group” means a group of atoms having at least 3 positions that are capable of forming covalent linkages to at least 3 groups. In certain embodiments, a branching group provides a plurality of reactive sites for connecting tethered ligands to an oligonucleotide via a conjugate linker and/or a cleavable moiety.

“Cell-targeting moiety” means a conjugate group or portion of a conjugate group that is capable of binding to a particular cell type or particular cell types.

“cEt” or “constrained ethyl” means a ribosyl bicyclic sugar moiety wherein the second ring of the bicyclic sugar is formed via a bridge connecting the 4′-carbon and the 2′-carbon, wherein the bridge has the formula: 4′-CH(CH₃)—O-2′, and wherein the methyl group of the bridge is in the S configuration.

“cEt nucleoside” means a nucleoside comprising a cEt modified sugar moiety.

“Chemical modification” in a compound describes the substitutions or changes through chemical reaction, of any of the units in the compound relative to the original state of such unit. “Modified nucleoside” means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase. “Modified oligonucleotide” means an oligonucleotide comprising at least one modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.

“Chemically distinct region” refers to a region of a compound that is in some way chemically different than another region of the same compound. For example, a region having 2′-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2′-O-methoxyethyl modifications.

“Chimeric antisense compounds” means antisense compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits.

“Cleavable bond” means any chemical bond capable of being split. In certain embodiments, a cleavable bond is selected from among: an amide, a polyamide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, a di-sulfide, or a peptide.

“Cleavable moiety” means a bond or group of atoms that is cleaved under physiological conditions, for example, inside a cell, an animal, or a human.

“Complementary” in reference to an oligonucleotide means the nucleobase sequence of such oligonucleotide or one or more regions thereof matches the nucleobase sequence of another oligonucleotide or nucleic acid or one or more regions thereof when the two nucleobase sequences are aligned in opposing directions. Nucleobase matches or complementary nucleobases, as described herein, are limited to the following pairs: adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methyl cytosine (^(m)C) and guanine (G) unless otherwise specified. Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside and may include one or more nucleobase mismatches. By contrast, “fully complementary” or “100% complementary” in reference to oligonucleotides means that such oligonucleotides have nucleobase matches at each nucleoside without any nucleobase mismatches.

“Conjugate group” means a group of atoms that is attached to an oligonucleotide. Conjugate groups include a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.

“Conjugate linker” means a group of atoms comprising at least one bond that connects a conjugate moiety to an oligonucleotide.

“Conjugate moiety” means a group of atoms that is attached to an oligonucleotide via a conjugate linker.

“Contiguous” in the context of an oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other. For example, “contiguous nucleobases” means nucleobases that are immediately adjacent to each other in a sequence.

“Designing” or “Designed to” refer to the process of designing a compound that specifically hybridizes with a selected nucleic acid molecule.

“Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, the diluent in an injected composition can be a liquid, e.g. saline solution.

“Differently modified” means chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2′-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2′-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.

“Dose” means a specified quantity of a compound or pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose may require a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual. In other embodiments, the compound or pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week or month.

“Dosing regimen” is a combination of doses designed to achieve one or more desired effects.

“Double-stranded antisense compound” means an antisense compound comprising two oligomeric compounds that are complementary to each other and form a duplex, and wherein one of the two said oligomeric compounds comprises an oligonucleotide.

“Effective amount” means the amount of compound sufficient to effectuate a desired physiological outcome in an individual in need of the compound. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.

“Efficacy” means the ability to produce a desired effect.

“Expression” includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to, the products of transcription and translation.

“Gapmer” means an oligonucleotide comprising an internal region having a plurality of nucleosides that support RNase H cleavage positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the “gap” and the external regions may be referred to as the “wings.”

“Hybridization” means the annealing of oligonucleotides and/or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an oligonucleotide and a nucleic acid target.

“Immediately adjacent” means there are no intervening elements between the immediately adjacent elements of the same kind (e.g. no intervening nucleobases between the immediately adjacent nucleobases).

“Individual” means a human or non-human animal selected for treatment or therapy.

“Inhibiting the expression or activity” refers to a reduction or blockade of the expression or activity relative to the expression of activity in an untreated or control sample and does not necessarily indicate a total elimination of expression or activity.

“Internucleoside linkage” means a group or bond that forms a covalent linkage between adjacent nucleosides in an oligonucleotide. “Modified internucleoside linkage” means any internucleoside linkage other than a naturally occurring, phosphate internucleoside linkage. Non-phosphate linkages are referred to herein as modified internucleoside linkages.

“Lengthened oligonucleotides” are those that have one or more additional nucleosides relative to an oligonucleotide disclosed herein, e.g. a parent oligonucleotide.

“Linked nucleosides” means adjacent nucleosides linked together by an internucleoside linkage.

“Linker-nucleoside” means a nucleoside that links an oligonucleotide to a conjugate moiety. Linker-nucleosides are located within the conjugate linker of a compound. Linker-nucleosides are not considered part of the oligonucleotide portion of a compound even if they are contiguous with the oligonucleotide.

“Mismatch” or “non-complementary” means a nucleobase of a first oligonucleotide that is not complementary to the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligonucleotides are aligned. For example, nucleobases including but not limited to a universal nucleobase, inosine, and hypoxanthine, are capable of hybridizing with at least one nucleobase but are still mismatched or non-complementary with respect to nucleobase to which it hybridized. As another example, a nucleobase of a first oligonucleotide that is not capable of hybridizing to the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligonucleotides are aligned is a mismatch or non-complementary nucleobase.

“Modulating” refers to changing or adjusting a feature in a cell, tissue, organ or organism. For example, modulating PNPLA3 RNA can mean to increase or decrease the level of PNPLA3 RNA and/or PNPLA3 protein in a cell, tissue, organ or organism. A “modulator” effects the change in the cell, tissue, organ or organism. For example, a PNPLA3 compound can be a modulator that decreases the amount of PNPLA3 RNA and/or PNPLA3 protein in a cell, tissue, organ or organism.

“MOE” means methoxyethyl.

“Monomer” refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides.

“Motif” means the pattern of unmodified and/or modified sugar moieties, nucleobases, and/or internucleoside linkages, in an oligonucleotide.

“Natural” or “naturally occurring” means found in nature.

“Non-bicyclic modified sugar” or “non-bicyclic modified sugar moiety” means a modified sugar moiety that comprises a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.

“Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, and double-stranded nucleic acids.

“Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid. As used herein a “naturally occurring nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). A “modified nucleobase” is a naturally occurring nucleobase that is chemically modified. A “universal base” or “universal nucleobase” is a nucleobase other than a naturally occurring nucleobase and modified nucleobase, and is capable of pairing with any nucleobase.

“Nucleobase sequence” means the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar or internucleoside linkage.

“Nucleoside” means a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each, independently, unmodified or modified. “Modified nucleoside” means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety. Modified nucleosides include abasic nucleosides, which lack a nucleobase.

“Oligomeric compound” means a compound comprising a single oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group.

“Oligonucleotide” means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another. Unless otherwise indicated, oligonucleotides consist of 8-80 linked nucleosides. “Modified oligonucleotide” means an oligonucleotide, wherein at least one sugar, nucleobase, or internucleoside linkage is modified. “Unmodified oligonucleotide” means an oligonucleotide that does not comprise any sugar, nucleobase, or internucleoside modification.

“Parent oligonucleotide” means an oligonucleotide whose sequence is used as the basis of design for more oligonucleotides of similar sequence but with different lengths, motifs, and/or chemistries. The newly designed oligonucleotides may have the same or overlapping sequence as the parent oligonucleotide.

“Parenteral administration” means administration through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration.

“Pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an individual. For example, a pharmaceutically acceptable carrier can be a sterile aqueous solution, such as PBS or water-for-injection.

“Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of compounds, such as oligomeric compounds or oligonucleotides, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

“Pharmaceutical agent” means a compound that provides a therapeutic benefit when administered to an individual.

“Pharmaceutical composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more compounds or salt thereof and a sterile aqueous solution.

“Phosphorothioate linkage” means a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. A phosphorothioate internucleoside linkage is a modified internucleoside linkage.

“Phosphorus moiety” means a group of atoms comprising a phosphorus atom. In certain embodiments, a phosphorus moiety comprises a mono-, di-, or tri-phosphate, or phosphorothioate.

“Portion” means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an oligomeric compound.

“Prevent” refers to delaying or forestalling the onset, development or progression of a disease, disorder, or condition for a period of time from minutes to indefinitely.

“Prodrug” means a compound in a form outside the body which, when administered to an individual, is metabolized to another form within the body or cells thereof. In certain embodiments, the metabolized form is the active, or more active, form of the compound (e.g., drug). Typically conversion of a prodrug within the body is facilitated by the action of an enzyme(s) (e.g., endogenous or viral enzyme) or chemical(s) present in cells or tissues, and/or by physiologic conditions.

“Reduce” means to bring down to a smaller extent, size, amount, or number.

“RefSeq No.” is a unique combination of letters and numbers assigned to a sequence to indicate the sequence is for a particular target transcript (e.g., target gene). Such sequence and information about the target gene (collectively, the gene record) can be found in a genetic sequence database. Genetic sequence databases include the NCBI Reference Sequence database, GenBank, the European Nucleotide Archive, and the DNA Data Bank of Japan (the latter three forming the International Nucleotide Sequence Database Collaboration or INSDC).

“Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.

“RNAi compound” means an antisense compound that acts, at least in part, through RISC or Ago2, but not through RNase H, to modulate a target nucleic acid and/or protein encoded by a target nucleic acid. RNAi compounds include, but are not limited to double-stranded siRNA, single-stranded RNA (ssRNA), and microRNA, including microRNA mimics.

“Segments” are defined as smaller or sub-portions of regions within a nucleic acid.

“Side effects” means physiological disease and/or conditions attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.

“Single-stranded” in reference to a compound means the compound has only one oligonucleotide. “Self-complementary” means an oligonucleotide that at least partially hybridizes to itself. A compound consisting of one oligonucleotide, wherein the oligonucleotide of the compound is self-complementary, is a single-stranded compound. A single-stranded compound may be capable of binding to a complementary compound to form a duplex.

“Sites” are defined as unique nucleobase positions within a target nucleic acid.

“Specifically hybridizable” refers to an oligonucleotide having a sufficient degree of complementarity between the oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids. In certain embodiments, specific hybridization occurs under physiological conditions.

“Specifically inhibit” with reference to a target nucleic acid means to reduce or block expression of the target nucleic acid while exhibiting fewer, minimal, or no effects on non-target nucleic acids. Reduction does not necessarily indicate a total elimination of the target nucleic acid's expression.

“Standard cell assay” means assay(s) described in the Examples and reasonable variations thereof.

“Standard in vivo experiment” means the procedure(s) described in the Example(s) and reasonable variations thereof.

“Stereorandom chiral center” in the context of a population of molecules of identical molecular formula means a chiral center having a random stereochemical configuration. For example, in a population of molecules comprising a stereorandom chiral center, the number of molecules having the (S) configuration of the stereorandom chiral center may be but is not necessarily the same as the number of molecules having the (R) configuration of the stereorandom chiral center. The stereochemical configuration of a chiral center is considered random when it is the result of a synthetic method that is not designed to control the stereochemical configuration. In certain embodiments, a stereorandom chiral center is a stereorandom phosphorothioate internucleoside linkage.

“Sugar moiety” means an unmodified sugar moiety or a modified sugar moiety. “Unmodified sugar moiety” or “unmodified sugar” means a 2′-OH(H) ribosyl moiety, as found in RNA (an “unmodified RNA sugar moiety”), or a 2′-H(H) moiety, as found in DNA (an “unmodified DNA sugar moiety”). “Modified sugar moiety” or “modified sugar” means a modified furanosyl sugar moiety or a sugar surrogate. “Modified furanosyl sugar moiety” means a furanosyl sugar comprising a non-hydrogen substituent in place of at least one hydrogen or hydroxyl of an unmodified sugar moiety. In certain embodiments, a modified furanosyl sugar moiety is a 2′-substituted sugar moiety. Such modified furanosyl sugar moieties include bicyclic sugars and non-bicyclic sugars.

“Sugar surrogate” means a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to another group, such as an internucleoside linkage, conjugate group, or terminal group in an oligonucleotide. Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to complementary compounds or nucleic acids.

“Synergy” or “synergize” refers to an effect of a combination that is greater than additive of the effects of each component alone at the same doses.

“PNPLA3” means any nucleic acid or protein of PNPLA3. “PNPLA3 nucleic acid” means any nucleic acid encoding PNPLA3. For example, in certain embodiments, a PNPLA3 nucleic acid includes a DNA sequence encoding PNPLA3, an RNA sequence transcribed from DNA encoding PNPLA3 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding PNPLA3. “PNPLA3 mRNA” means an mRNA encoding a PNPLA3 protein. The target may be referred to in either upper or lower case.

“PNPLA3 specific inhibitor” refers to any agent capable of specifically inhibiting PNPLA3 RNA and/or PNPLA3 protein expression or activity at the molecular level. For example, PNPLA3 specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of PNPLA3 RNA and/or PNPLA3 protein.

“Target gene” refers to a gene encoding a target.

“Targeting” means the specific hybridization of a compound to a target nucleic acid in order to induce a desired effect.

“Target nucleic acid,” “target RNA,” “target RNA transcript” and “nucleic acid target” all mean a nucleic acid capable of being targeted by compounds described herein.

“Target region” means a portion of a target nucleic acid to which one or more compounds is targeted.

“Target segment” means the sequence of nucleotides of a target nucleic acid to which a compound is targeted. “5′ target site” refers to the 5′-most nucleotide of a target segment. “3′ target site” refers to the 3′-most nucleotide of a target segment.

“Terminal group” means a chemical group or group of atoms that is covalently linked to a terminus of an oligonucleotide.

“Therapeutically effective amount” means an amount of a compound, pharmaceutical agent, or composition that provides a therapeutic benefit to an individual.

“Treat” refers to administering a compound or pharmaceutical composition to an animal in order to effect an alteration or improvement of a disease, disorder, or condition in the animal.

Certain Embodiments

Certain embodiments provide methods, compounds and compositions for inhibiting PNPLA3 (PNPLA3) expression.

Certain embodiments provide compounds targeted to a PNPLA3 nucleic acid. In certain embodiments, the PNPLA3 nucleic acid has the sequence set forth in RefSeq or GENBANK Accession No. NM_025225.2 (incorporated by reference, disclosed herein as SEQ ID NO: 1); NC_000022.11 truncated from nucleotides 43921001 to U.S. Pat. No. 43,954,500 (incorporated by reference, disclosed herein as SEQ ID NO: 2); AK123806.1 (incorporated by reference, disclosed herein as SEQ ID NO: 3); BQ686328.1 (incorporated by reference, disclosed herein as SEQ ID NO: 4); BF762711.1 (incorporated by reference, disclosed herein as SEQ ID NO: 5); DA290491.1 (incorporated by reference, disclosed herein as SEQ ID NO: 6); and the sequences listed as SEQ ID Nos 7, 8, 9, and 10. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded.

In certain embodiments, the compound comprises a modified oligonucleotide 16 linked nucleosides in length. In certain embodiments, the compound is an antisense compound or oligomeric compound.

Certain embodiments provide a compound comprising a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded. In certain embodiments, the modified oligonucleotide is 16 to 30 linked nucleosides in length.

Certain embodiments provide a compound comprising a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded.

Certain embodiments provide a compound comprising a modified oligonucleotide 12 to 30 linked nucleosides in length and complementary within nucleobases 5567-5642, 5644-5731, 5567-5731, 5567-5620, 13697-13733, 20553-20676, 20664-20824, 20553-20824, and 25844-25912 of SEQ ID NO: 2, wherein said modified oligonucleotide is at least 85%, at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 2. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded. In certain embodiments, the modified oligonucleotide is 16 to 30 linked nucleosides in length.

In certain embodiments, compounds target nucleotides 5567-5620 of a PNPLA3 nucleic acid. In certain embodiments, compounds target within nucleotides 5567-5642, 5644-5731, 5567-5731, 5567-5620 of a PNPLA3 nucleic acid having the nucleobase sequence of SEQ ID NO: 2. In certain embodiments, compounds have at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion complementary to an equal length portion within nucleotides 5567-5642, 5644-5731, 5567-5731, 5567-5620 of a PNPLA3 nucleic acid having the nucleobase sequence of SEQ ID NO: 2. In certain embodiments, these compounds are antisense compounds, oligomeric compounds, or oligonucleotides.

In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the modified oligonucleotide is 16 to 30 linked nucleosides in length.

In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the modified oligonucleotide is 16 to 30 linked nucleosides in length.

In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899.

In certain embodiments, compounds targeted to PNPLA3 is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. Out of over 2,384 compounds that were screened as described in the Examples section below, ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, and 975612 emerged as the top lead compounds.

In certain embodiments, any of the foregoing modified oligonucleotides comprises at least one modified internucleoside linkage, at least one modified sugar, and/or at least one modified nucleobase.

In certain embodiments, any of the foregoing modified oligonucleotides comprises at least one modified sugar. In certain embodiments, at least one modified sugar comprises a 2′-O-methoxyethyl group. In certain embodiments, at least one modified sugar is a bicyclic sugar, such as a 4′-CH(CH3)-O-2′ group, a 4′-CH2-O-2′ group, or a 4′-(CH2)2-O-2′group.

In certain embodiments, the modified oligonucleotide comprises at least one modified internucleoside linkage, such as a phosphorothioate internucleoside linkage.

In certain embodiments, any of the foregoing modified oligonucleotides comprises at least one modified nucleobase, such as 5-methylcytosine.

In certain embodiments, any of the foregoing modified oligonucleotides comprises:

-   -   a gap segment consisting of linked deoxynucleosides;     -   a 5′ wing segment consisting of linked nucleosides; and     -   a 3′ wing segment consisting of linked nucleosides;         wherein the gap segment is positioned between the 5′ wing         segment and the 3′ wing segment and wherein each nucleoside of         each wing segment comprises a modified sugar. In certain         embodiments, the modified oligonucleotide is 12 to 30 linked         nucleosides in length having a nucleobase sequence comprising         the sequence recited in any one of SEQ ID NOs: 1089, 1757, 141,         1982, 330, 1665, 408, 830, and 899. In certain embodiments, the         modified oligonucleotide is 16 to 30 linked nucleosides in         length having a nucleobase sequence comprising the sequence         recited in any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330,         1665, 408, 830, and 899. In certain embodiments, the modified         oligonucleotide is 16 linked nucleosides in length having a         nucleobase sequence consisting of the sequence recited in any         one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830,         and 899.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide 12-30 linked nucleobases in length having a nucleobase sequence comprising the sequence recited in any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899, wherein the modified oligonucleotide comprises

-   -   a gap segment consisting of ten linked deoxynucleosides;     -   a 5′ wing segment consisting of three linked nucleosides; and     -   a 3′ wing segment consisting of three linked nucleosides;         wherein the gap segment is positioned between the 5′ wing         segment and the 3′ wing segment, wherein each nucleoside of each         wing segment comprises a cEt sugar; wherein each internucleoside         linkage is a phosphorothioate linkage and wherein each cytosine         is a 5-methylcytosine. In certain embodiments, the modified         oligonucleotide consists of 16-30 linked nucleosides. In certain         embodiments, the modified oligonucleotide consists of 16 linked         nucleosides.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide, wherein the modified oligonucleotide is 16 linked nucleosides in length and consists of the sequence of SEQ ID NO: 1089, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of ten linked deoxynucleosides;     -   a 5′ wing segment consisting of three linked nucleosides; and     -   a 3′ wing segment consisting of three linked nucleosides;     -   wherein the gap segment is positioned between the 5′ wing         segment and the 3′ wing segment, wherein each nucleoside of each         wing segment comprises a cEt sugar; wherein each internucleoside         linkage is a phosphorothioate linkage; and wherein each cytosine         is a 5-methylcytosine.

In certain embodiments, a compound consists of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide is 16 linked nucleosides in length and consists of the sequence of SEQ ID NO: 1089, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of ten linked deoxynucleosides;     -   a 5′ wing segment consisting of three linked nucleosides; and     -   a 3′ wing segment consisting of three linked nucleosides;     -   wherein the gap segment is positioned between the 5′ wing         segment and the 3′ wing segment, wherein each nucleoside of each         wing segment comprises a cEt sugar; wherein each internucleoside         linkage is a phosphorothioate linkage; wherein each cytosine is         a 5-methylcytosine; and wherein the conjugate group is         positioned at the 5′end of the modified oligonucleotide and is

In certain embodiments, a compound comprises or consists of ION 916333 or salt thereof, having the following chemical structure (SEQ ID NO: 1089):

In certain embodiments, a compound comprises or consists of ION 975616 or salt thereof, having the following chemical structure (SEQ ID NO: 1089):

In certain embodiments, a compound comprises or consists of the sodium salt of 975616, having the following chemical structure (SEQ ID NO: 1089):

In certain embodiments, a compound comprises or consists of ION 975613 or salt thereof, having the following chemical structure (SEQ ID NO: 330):

In certain embodiments, a compound comprises or consists of the sodium salt of 975613, having the following chemical structure (SEQ ID NO: 330):

In certain embodiments, a compound comprises or consists of ION 975612 or salt thereof, having the following chemical structure (SEQ ID NO: 899):

In certain embodiments, a compound comprises or consists of the sodium salt of 975612, having the following chemical structure (SEQ ID NO: 899):

In certain embodiments, a compound comprises or consists of ION 916789 or salt thereof, having the following chemical structure (SEQ ID NO: 899):

In certain embodiments, a compound comprises or consists of the sodium salt of 916789, having the following chemical structure (SEQ ID NO: 330):

In certain embodiments, a compound comprises or consists of the sodium salt of 916789, having the following chemical structure (SEQ ID NO: 330):

In certain embodiments, a compound comprises or consists of the sodium salt of 916602, having the following chemical structure (SEQ ID NO: 330):

In any of the foregoing embodiments, the compound or oligonucleotide can be at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 1000% complementary to a nucleic acid encoding PNPLA3.

In any of the foregoing embodiments, the compound can be single-stranded. In certain embodiments, the compound comprises deoxyribonucleotides. In certain embodiments, the compound is double-stranded. In certain embodiments, the compound is double-stranded and comprises ribonucleotides. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound.

In any of the foregoing embodiments, the compound can be 8 to 80, 10 to 30, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked nucleosides in length. In certain embodiments, the compound comprises or consists of an oligonucleotide.

In certain embodiments, a compound comprises a modified oligonucleotide described herein and a conjugate group. In certain embodiments, the conjugate group is linked to the modified oligonucleotide at the 5′ end of the modified oligonucleotide. In certain embodiments, the conjugate group is linked to the modified oligonucleotide at the 3′ end of the modified oligonucleotide. In certain embodiments, the conjugate group comprises at least one N-Acetylgalactosamine (GalNAc), at least two N-Acetylgalactosamines (GalNAcs), or at least three N-Acetylgalactosamines (GalNAcs).

In certain embodiments, compounds or compositions provided herein comprise a pharmaceutically acceptable salt of the modified oligonucleotide. In certain embodiments, the salt is a sodium salt. In certain embodiments, the salt is a potassium salt.

In certain embodiments, the compounds or compositions as described herein are active by virtue of having at least one of an in vitro IC₅₀ of less than 2 μM, less than 1.5 μM, less than 1 μM, less than 0.9 μM, less than 0.8 μM, less than 0.7 μM, less than 0.6 μM, less than 0.5 μM, less than 0.4 μM, less than 0.3 μM, less than 0.2 μM, less than 0.1 μM, less than 0.05 μM, less than 0.04 μM, less than 0.03 μM, less than 0.02 μM, or less than 0.01 μM.

In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having at least one of an increase in alanine transaminase (ALT) or aspartate transaminase (AST) value of no more than 4 fold, 3 fold, or 2 fold over control animals, or an increase in liver, spleen, or kidney weight of no more than 30%, 20%, 15%, 12%, 10%, 5%, or 2% compared to control animals. In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having no increase of ALT or AST over control animals. In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having no increase in liver, spleen, or kidney weight over control animals.

Certain embodiments provide a composition comprising the compound of any of the aforementioned embodiments or any pharmaceutically acceptable salt thereof and at least one of a pharmaceutically acceptable carrier or diluent. In certain embodiments, the composition has a viscosity less than about 40 centipoise (cP), less than about 30 centipose (cP), less than about 20 centipose (cP), less than about 15 centipose (cP), or less than about 10 centipose (cP). In certain embodiments, the composition having any of the aforementioned viscosities comprises a compound provided herein at a concentration of about 100 mg/mL, about 125 mg/mL, about 150 mg/mL, about 175 mg/mL, about 200 mg/mL, about 225 mg/mL, about 250 mg/mL, about 275 mg/mL, or about 300 mg/mL. In certain embodiments, the composition having any of the aforementioned viscosities and/or compound concentrations has a temperature of room temperature, or about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., or about 30° C.

Certain Indications

Certain embodiments provided herein relate to methods of inhibiting PNPLA3 expression, which can be useful for treating, preventing, or ameliorating a disease associated with PNPLA3 in an individual, by administration of a compound that targets PNPLA3. In certain embodiments, the compound can be a PNPLA3 specific inhibitor. In certain embodiments, the compound can be an antisense compound, an oligomeric compound, or an oligonucleotide targeted to PNPLA3.

Examples of diseases associated with PNPLA3 treatable, preventable, and/or ameliorable with the methods provided herein include liver disease, NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. Certain compounds provided herein are directed to compounds and compositions that reduce liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation in an animal.

In certain embodiments, a method of treating, preventing, or ameliorating a disease associated with PNPLA3 in an individual comprises administering to the individual a compound comprising a PNPLA3 specific inhibitor, thereby treating, preventing, or ameliorating the disease. In certain embodiments, the individual is identified as having, or at risk of having, a disease associated with PNPLA3. In certain embodiments, the disease is a liver disease. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound. In certain embodiments, the compound is administered to the individual parenterally. In certain embodiments, administering the compound improves, preserves, or prevents liver damage, steatosis, liver fibrosis, cirrhosis, elevated transaminases, or hepatic fat accumulation in an animal.

In certain embodiments, a method of treating, preventing, or ameliorating liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation in an animal comprises administering to the individual a compound comprising a PNPLA3 specific inhibitor, thereby treating, preventing, or ameliorating liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound. In certain embodiments, the compound is administered to the individual parenterally. In certain embodiments, administering the compound improves, preserves, or prevents liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation. In certain embodiments, the individual is identified as having, or at risk of having, a disease associated with PNPLA3.

In certain embodiments, a method of inhibiting expression of PNPLA3 in an individual having, or at risk of having, a disease associated with PNPLA3 comprises administering to the individual a compound comprising a PNPLA3 specific inhibitor, thereby inhibiting expression of PNPLA3 in the individual. In certain embodiments, administering the compound inhibits expression of PNPLA3 in the liver. In certain embodiments, the disease is a liver disease. In certain embodiments, the individual has, or is at risk of having, NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. In certain embodiments, the individual has, or is at risk of having, liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound. In certain embodiments, the compound is administered to the individual parenterally. In certain embodiments, administering the compound improves, preserves, or prevents liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation.

In certain embodiments, a method of inhibiting expression of PNPLA3 in a cell comprises contacting the cell with a compound comprising a PNPLA3 specific inhibitor, thereby inhibiting expression of PNPLA3 in the cell. In certain embodiments, the cell is a hepatocyte. In certain embodiments, the cell is in the liver. In certain embodiments, the cell is in the liver of an individual who has, or is at risk of having, liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound.

In certain embodiments, a method of reducing or inhibiting liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation in an individual having, or at risk of having, a disease associated with PNPLA3 comprises administering to the individual a compound comprising a PNPLA3 specific inhibitor, thereby reducing or inhibiting liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation in the individual. In certain embodiments, the individual has, or is at risk of having, NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound. In certain embodiments, the compound is administered to the individual parenterally. In certain embodiments, the individual is identified as having, or at risk of having, a disease associated with PNPLA3.

Certain embodiments are drawn to a compound comprising a PNPLA3 specific inhibitor for use in treating a disease associated with PNPLA3. In certain embodiments, the disease is NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound. In certain embodiments, the compound is administered to the individual parenterally.

Certain embodiments are drawn to a compound comprising a PNPLA3 specific inhibitor for use in reducing or inhibiting liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation in an individual having, or at risk of having, NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound.

Certain embodiments are drawn to the use of a compound comprising a PNPLA3 specific inhibitor for the manufacture or preparation of a medicament for treating a disease associated with PNPLA3. Certain embodiments are drawn to the use of a compound comprising a PNPLA3 specific inhibitor for the preparation of a medicament for treating a disease associated with PNPLA3. In certain embodiments, the disease is a liver disease. In certain embodiments, the disease is NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or an oligomeric compound.

Certain embodiments are drawn to the use of a compound comprising a PNPLA3 specific inhibitor for the manufacture or preparation of a medicament for reducing or inhibiting liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation in an individual having, or at risk of having, a liver disease associated with PNPLA3. In certain embodiments, the liver disease is NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. Certain embodiments are drawn to use of a compound comprising a PNPLA3 specific inhibitor for the preparation of a medicament for treating a disease associated with PNPLA3. In certain embodiments, the disease is NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or an oligomeric compound.

In any of the foregoing methods or uses, the compound can be targeted to PNPLA3. In certain embodiments, the compound comprises or consists of a modified oligonucleotide, for example, a modified oligonucleotide 8 to 80 linked nucleosides in length, 10 to 30 linked nucleosides in length, 12 to 30 linked nucleosides in length, or 20 linked nucleosides in length. In certain embodiments, the modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to any of the nucleobase sequences recited in SEQ ID NOs: 1-10. In certain embodiments, the modified oligonucleotide comprises at least one modified internucleoside linkage, at least one modified sugar and/or at least one modified nucleobase. In certain embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage, the modified sugar is a bicyclic sugar or a 2′-O-methoxyethyl modified sugar, and the modified nucleobase is a 5-methylcytosine. In certain embodiments, the modified oligonucleotide comprises a gap segment consisting of linked deoxynucleosides; a 5′ wing segment consisting of linked nucleosides; and a 3′ wing segment consisting of linked nucleosides, wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

In any of the foregoing embodiments, the modified oligonucleotide is 12 to 30, 15 to 30, 15 to 25, 15 to 24, 16 to 24, 17 to 24, 18 to 24, 19 to 24, 20 to 24, 19 to 22, 20 to 22, 16 to 20, or 16 or 20 linked nucleosides in length. In certain embodiments, the modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to any of the nucleobase sequences recited in SEQ ID NOs: 1-10.

In any of the foregoing methods or uses, the compound comprises or consists of a modified oligonucleotide 16 to 30 linked nucleosides in length and having a nucleobase sequence comprising any one of SEQ ID NOs: 17-2169, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of linked 2′-deoxynucleosides;     -   a 5′ wing segment consisting of linked nucleosides; and     -   a 3′ wing segment consisting of linked nucleosides;         wherein the gap segment is positioned between the 5′ wing         segment and the 3′ wing segment and wherein each nucleoside of         each wing segment comprises a modified sugar.

In any of the foregoing methods or uses, the compound comprises or consists a modified oligonucleotide 16 linked nucleosides in length having a nucleobase sequence comprising the sequence recited in any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899, wherein the modified oligonucleotide comprises

-   -   a gap segment consisting of ten linked deoxynucleosides;     -   a 5′ wing segment consisting of three linked nucleosides; and a         3′ wing segment consisting of three linked nucleosides;         wherein the gap segment is positioned between the 5′ wing         segment and the 3′ wing segment, wherein each nucleoside of each         wing segment comprises a cEt sugar; wherein each internucleoside         linkage is a phosphorothioate linkage and wherein each cytosine         is a 5-methylcytosine. In certain embodiments, the modified         oligonucleotide is 16-30 linked nucleosides in length.

In certain embodiments, a compound comprises or consists of ION 916333 or salt thereof, having the following chemical structure (SEQ ID NO: 1089):

In certain embodiments, a compound comprises or consists of ION 975616 or salt thereof, having the following chemical structure (SEQ ID NO: 1089):

In certain embodiments, a compound comprises or consists of the sodium salt of 975616, having the following chemical structure (SEQ ID NO: 1089):

In certain embodiments, a compound comprises or consists of ION 975613 or salt thereof, having the following chemical structure (SEQ ID NO: 330):

In certain embodiments, a compound comprises or consists of the sodium salt of 975613, having the following chemical structure (SEQ ID NO: 330):

In certain embodiments, a compound comprises or consists of ION 975612 or salt thereof, having the following chemical structure (SEQ ID NO: 899):

In certain embodiments, a compound comprises or consists of the sodium salt of 975612, having the following chemical structure (SEQ ID NO: 899):

In certain embodiments, a compound comprises or consists of ION 916789 or salt thereof, having the following chemical structure (SEQ ID NO: 899):

In certain embodiments, a compound comprises or consists of the sodium salt of 916789, having the following chemical structure (SEQ ID NO: 899):

In certain embodiments, a compound comprises or consists of ION 916602 or salt thereof, having the following chemical structure (SEQ ID NO: 330):

In certain embodiments, a compound comprises or consists of the sodium salt of 916602, having the following chemical structure (SEQ ID NO: 330):

In any of the foregoing methods or uses, the compound can be administered parenterally. For example, in certain embodiments the compound can be administered through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration.

Certain Compounds

In certain embodiments, compounds described herein can be antisense compounds. In certain embodiments, the antisense compound comprises or consists of an oligomeric compound. In certain embodiments, the oligomeric compound comprises a modified oligonucleotide. In certain embodiments, the modified oligonucleotide has a nucleobase sequence complementary to that of a target nucleic acid.

In certain embodiments, a compound described herein comprises or consists of a modified oligonucleotide. In certain embodiments, the modified oligonucleotide has a nucleobase sequence complementary to that of a target nucleic acid.

In certain embodiments, a compound or antisense compound is single-stranded. Such a single-stranded compound or antisense compound comprises or consists of an oligomeric compound. In certain embodiments, such an oligomeric compound comprises or consists of an oligonucleotide and optionally a conjugate group. In certain embodiments, the oligonucleotide is an antisense oligonucleotide. In certain embodiments, the oligonucleotide is modified. In certain embodiments, the oligonucleotide of a single-stranded antisense compound or oligomeric compound comprises a self-complementary nucleobase sequence.

In certain embodiments, compounds are double-stranded. Such double-stranded compounds comprise a first modified oligonucleotide having a region complementary to a target nucleic acid and a second modified oligonucleotide having a region complementary to the first modified oligonucleotide. In certain embodiments, the modified oligonucleotide is an RNA oligonucleotide. In such embodiments, the thymine nucleobase in the modified oligonucleotide is replaced by a uracil nucleobase. In certain embodiments, compound comprises a conjugate group. In certain embodiments, one of the modified oligonucleotides is conjugated. In certain embodiments, both the modified oligonucleotides are conjugated. In certain embodiments, the first modified oligonucleotide is conjugated. In certain embodiments, the second modified oligonucleotide is conjugated. In certain embodiments, the first modified oligonucleotide is 16-30 linked nucleosides in length and the second modified oligonucleotide is 16-30 linked nucleosides in length. In certain embodiments, one of the modified oligonucleotides has a nucleobase sequence comprising at least 8 contiguous nucleobases of any of SEQ ID NOs: 17-2169.

In certain embodiments, antisense compounds are double-stranded. Such double-stranded antisense compounds comprise a first oligomeric compound having a region complementary to a target nucleic acid and a second oligomeric compound having a region complementary to the first oligomeric compound. The first oligomeric compound of such double stranded antisense compounds typically comprises or consists of a modified oligonucleotide and optionally a conjugate group. The oligonucleotide of the second oligomeric compound of such a double-stranded antisense compound may be modified or unmodified. Either or both oligomeric compounds of a double-stranded antisense compound may comprise a conjugate group. The oligomeric compounds of double-stranded antisense compounds may include non-complementary overhanging nucleosides.

Examples of single-stranded and double-stranded compounds include, but are not limited to, oligonucleotides, siRNAs, microRNA targeting oligonucleotides, and single-stranded RNAi compounds, such as small hairpin RNAs (shRNAs), single-stranded siRNAs (ssRNAs), and microRNA mimics.

In certain embodiments, a compound described herein has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.

In certain embodiments, a compound described herein comprises an oligonucleotide 12 to 30 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 12 to 22 linked subunits in length. In certain embodiments, compound described herein comprises an oligonucleotide 14 to 30 linked subunits in length. In certain embodiments, compound described herein comprises an oligonucleotide 14 to 20 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 15 to 30 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 15 to 20 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 16 to 30 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 16 to 20 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 17 to 30 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 17 to 20 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 18 to 30 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 18 to 20 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 20 to 30 linked subunits in length. In other words, such oligonucleotides are 12 to 30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30 subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20 subunits, 17 to 30 subunits, 17 to 20 subunits, 18 to 30 subunits, 18 to 20 subunits, or 20 to 30 subunits in length, respectively. In certain embodiments, a compound described herein comprises an oligonucleotide 14 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 16 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 17 linked subunits in length. In certain embodiments, compound described herein comprises an oligonucleotide 18 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 19 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 20 linked subunits in length. In other embodiments, a compound described herein comprises an oligonucleotide 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such embodiments, the compound described herein comprises an oligonucleotide 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 linked subunits in length, or a range defined by any two of the above values. In some embodiments the linked subunits are nucleotides, nucleosides, or nucleobases.

In certain embodiments, the compound may further comprise additional features or elements, such as a conjugate group, that are attached to the oligonucleotide. In certain embodiments, such compounds are antisense compounds. In certain embodiments, such compounds are oligomeric compounds. In embodiments where a conjugate group comprises a nucleoside (i.e. a nucleoside that links the conjugate group to the oligonucleotide), the nucleoside of the conjugate group is not counted in the length of the oligonucleotide.

In certain embodiments, compounds may be shortened or truncated. For example, a single subunit may be deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated compound targeted to a PNPLA3 nucleic acid may have two subunits deleted from the 5′ end, or alternatively, may have two subunits deleted from the 3′ end of the compound. Alternatively, the deleted nucleosides may be dispersed throughout the compound.

When a single additional subunit is present in a lengthened compound, the additional subunit may be located at the 5′ or 3′ end of the compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in a compound having two subunits added to the 5′ end (5′ addition), or alternatively, to the 3′ end (3′ addition) of the compound. Alternatively, the added subunits may be dispersed throughout the compound.

It is possible to increase or decrease the length of a compound, such as an oligonucleotide, and/or introduce mismatch bases without eliminating activity (Woolf et al. Proc. Natl. Acad. Sci. USA 1992, 89:7305-7309; Gautschi et al. J Natl. Cancer Inst. March 2001, 93:463-471; Maher and Dolnick Nuc. Acid. Res. 1998, 16:3341-3358). However, seemingly small changes in oligonucleotide sequence, chemistry and motif can make large differences in one or more of the many properties required for clinical development (Seth et al. J Med. Chem. 2009, 52, 10; Egli et al. J Am. Chem. Soc. 2011, 133, 16642).

In certain embodiments, compounds described herein are interfering RNA compounds (RNAi), which include double-stranded RNA compounds (also referred to as short-interfering RNA or siRNA) and single-stranded RNAi compounds (or ssRNA). Such compounds work at least in part through the RISC pathway to degrade and/or sequester a target nucleic acid (thus, include microRNA/microRNA-mimic compounds). As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence-specific RNAi, for example, short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term “RNAi” is meant to be equivalent to other terms used to describe sequence-specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics.

In certain embodiments, a compound described herein can comprise any of the oligonucleotide sequences targeted to PNPLA3 described herein. In certain embodiments, the compound can be double-stranded. In certain embodiments, the compound comprises a first strand comprising at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion of any one of SEQ ID NOs: 17-2169 and a second strand. In certain embodiments, the compound comprises a first strand comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169 and a second strand. In certain embodiments, the compound comprises ribonucleotides in which the first strand has uracil (U) in place of thymine (T) in any one of SEQ ID NOs: 17-2169. In certain embodiments, the compound comprises (i) a first strand comprising a nucleobase sequence complementary to the site on PNPLA3 to which any of SEQ ID NOs: 17-2169 is targeted, and (ii) a second strand. In certain embodiments, the compound comprises one or more modified nucleotides in which the 2′ position of the sugar contains a halogen (such as fluorine group; 2′-F) or contains an alkoxy group (such as a methoxy group; 2′-OMe). In certain embodiments, the compound comprises at least one 2′-F sugar modification and at least one 2′-OMe sugar modification. In certain embodiments, the at least one 2′-F sugar modification and at least one 2′-OMe sugar modification are arranged in an alternating pattern for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases along a strand of the dsRNA compound. In certain embodiments, the compound comprises one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages. The compounds may also be chemically modified nucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In other embodiments, the compound contains one or two capped strands, as disclosed, for example, by WO 00/63364, filed Apr. 19, 2000.

In certain embodiments, the first strand of the compound is an siRNA guide strand and the second strand of the compound is an siRNA passenger strand. In certain embodiments, the second strand of the compound is complementary to the first strand. In certain embodiments, each strand of the compound is 16, 17, 18, 19, 20, 21, 22, or 23 linked nucleosides in length. In certain embodiments, the first or second strand of the compound can comprise a conjugate group.

In certain embodiments, a compound described herein can comprise any of the oligonucleotide sequences targeted to PNPLA3 described herein. In certain embodiments, the compound is single stranded. In certain embodiments, such a compound is a single-stranded RNAi (ssRNAi) compound. In certain embodiments, the compound comprises at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion of any one of SEQ ID NOs: 17-2169. In certain embodiments, the compound comprises the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, the compound comprises ribonucleotides in which uracil (U) is in place of thymine (T) in any one of SEQ ID NOs: 17-2169. In certain embodiments, the compound comprises a nucleobase sequence complementary to the site on PNPLA3 to which any of SEQ ID NOs: 17-2169 is targeted. In certain embodiments, the compound comprises one or more modified nucleotides in which the 2′ position in the sugar contains a halogen (such as fluorine group; 2′-F) or contains an alkoxy group (such as a methoxy group; 2′-OMe). In certain embodiments, the compound comprises at least one 2′-F sugar modification and at least one 2′-OMe sugar modification. In certain embodiments, the at least one 2′-F sugar modification and at least one 2′-OMe sugar modification are arranged in an alternating pattern for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases along a strand of the compound. In certain embodiments, the compound comprises one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages. The compounds may also be chemically modified nucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In other embodiments, the compound contains a capped strand, as disclosed, for example, by WO 00/63364, filed Apr. 19, 2000. In certain embodiments, the compound consists of 16, 17, 18, 19, 20, 21, 22, or 23 linked nucleosides. In certain embodiments, the compound can comprise a conjugate group.

Certain Mechanisms

In certain embodiments, compounds described herein comprise or consist of modified oligonucleotides. In certain embodiments, compounds described herein are antisense compounds. In certain embodiments, compounds comprise oligomeric compounds. In certain embodiments, compounds described herein are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, compounds described herein selectively affect one or more target nucleic acid. Such compounds comprise a nucleobase sequence that hybridizes to one or more target nucleic acid, resulting in one or more desired antisense activity and does not hybridize to one or more non-target nucleic acid or does not hybridize to one or more non-target nucleic acid in such a way that results in a significant undesired antisense activity.

In certain antisense activities, hybridization of a compound described herein to a target nucleic acid results in recruitment of a protein that cleaves the target nucleic acid. For example, certain compounds described herein result in RNase H mediated cleavage of the target nucleic acid. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. The DNA in such an RNA:DNA duplex need not be unmodified DNA. In certain embodiments, compounds described herein are sufficiently “DNA-like” to elicit RNase H activity. Further, in certain embodiments, one or more non-DNA-like nucleoside in the gap of a gapmer is tolerated.

In certain antisense activities, compounds described herein or a portion of the compound is loaded into an RNA-induced silencing complex (RISC), ultimately resulting in cleavage of the target nucleic acid. For example, certain compounds described herein result in cleavage of the target nucleic acid by Argonaute. Compounds that are loaded into RISC are RNAi compounds. RNAi compounds may be double-stranded (siRNA) or single-stranded (ssRNA).

In certain embodiments, hybridization of compounds described herein to a target nucleic acid does not result in recruitment of a protein that cleaves that target nucleic acid. In certain such embodiments, hybridization of the compound to the target nucleic acid results in alteration of splicing of the target nucleic acid. In certain embodiments, hybridization of the compound to a target nucleic acid results in inhibition of a binding interaction between the target nucleic acid and a protein or other nucleic acid. In certain such embodiments, hybridization of the compound to a target nucleic acid results in alteration of translation of the target nucleic acid.

Antisense activities may be observed directly or indirectly. In certain embodiments, observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid, a change in the ratio of splice variants of a nucleic acid or protein, and/or a phenotypic change in a cell or animal.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

In certain embodiments, compounds described herein comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from an mRNA and a pre-mRNA, including intronic, exonic and untranslated regions. In certain embodiments, the target RNA is an mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain such embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron.

Nucleotide sequences that encode PNPLA3 include, without limitation, the following: RefSeq or GENBANK Accession Nos. NM_025225.2 (incorporated by reference, disclosed herein as SEQ ID NO: 1); GENBANK Accession No. NC_000022.11 truncated from nucleotides 43921001 to U.S. Pat. No. 43,954,500 (incorporated by reference, disclosed herein as SEQ ID NO: 2); AK123806.1 (incorporated by reference, disclosed herein as SEQ ID NO: 3); BQ686328.1 (incorporated by reference, disclosed herein as SEQ ID NO: 4); BF762711.1 (incorporated by reference, disclosed herein as SEQ ID NO: 5); DA290491.1 (incorporated by reference, disclosed herein as SEQ ID NO: 6); and the sequences listed as SEQ ID Nos. 7, 8, 9, and 10.

Hybridization

In some embodiments, hybridization occurs between a compound disclosed herein and a PNPLA3 nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Hybridization conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the compounds provided herein are specifically hybridizable with a PNPLA3 nucleic acid.

Complementarity

An oligonucleotide is said to be complementary to another nucleic acid when the nucleobase sequence of such oligonucleotide or one or more regions thereof matches the nucleobase sequence of another oligonucleotide or nucleic acid or one or more regions thereof when the two nucleobase sequences are aligned in opposing directions. Nucleobase matches or complementary nucleobases, as described herein, are limited to the following pairs: adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methyl cytosine (mC) and guanine (G), unless otherwise specified. Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside and may include one or more nucleobase mismatches. An oligonucleotide is fully complementary or 100% complementary when such oligonucleotides have nucleobase matches at each nucleoside without any nucleobase mismatches.

In certain embodiments, compounds described herein comprise or consist of modified oligonucleotides. In certain embodiments, compounds described herein are antisense compounds. In certain embodiments, compounds comprise oligomeric compounds. Non-complementary nucleobases between a compound and a PNPLA3 nucleic acid may be tolerated provided that the compound remains able to specifically hybridize to a target nucleic acid. Moreover, a compound may hybridize over one or more segments of a PNPLA3 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).

In certain embodiments, the compounds provided herein, or a specified portion thereof are at least, or are up to 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a PNPLA3 nucleic acid, a target region, target segment, or specified portion thereof. In certain embodiments, the compounds provided herein, or a specified portion thereof, are 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, 95% to 100%, or any number in between these ranges, complementary to a PNPLA3 nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of a compound with a target nucleic acid can be determined using routine methods.

For example, a compound in which 18 of 20 nucleobases of the compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining non-complementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, a compound which is 18 nucleobases in length having four non-complementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid. Percent complementarity of a compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).

In certain embodiments, compounds described herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, a compound may be fully complementary to a PNPLA3 nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of a compound is complementary to the corresponding nucleobase of a target nucleic acid. For example, a 20 nucleobase compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the compound. “Fully complementary” can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase compound is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the compound. At the same time, the entire 30 nucleobase compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the compound are also complementary to the target sequence.

In certain embodiments, compounds described herein comprise one or more mismatched nucleobases relative to the target nucleic acid. In certain such embodiments, antisense activity against the target is reduced by such mismatch, but activity against a non-target is reduced by a greater amount. Thus, in certain such embodiments, selectivity of the compound is improved. In certain embodiments, the mismatch is specifically positioned within an oligonucleotide having a gapmer motif. In certain such embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, or 8 from the 5′-end of the gap region. In certain such embodiments, the mismatch is at position 9, 8, 7, 6, 5, 4, 3, 2, 1 from the 3′-end of the gap region. In certain such embodiments, the mismatch is at position 1, 2, 3, or 4 from the 5′-end of the wing region. In certain such embodiments, the mismatch is at position 4, 3, 2, or 1 from the 3′-end of the wing region. In certain embodiments, the mismatch is specifically positioned within an oligonucleotide not having a gapmer motif. In certain such embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 5′-end of the oligonucleotide. In certain such embodiments, the mismatch is at position, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 3′-end of the oligonucleotide.

The location of a non-complementary nucleobase may be at the 5′ end or 3′ end of the compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer oligonucleotide.

In certain embodiments, compounds described herein that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a PNPLA3 nucleic acid, or specified portion thereof.

In certain embodiments, compounds described herein that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a PNPLA3 nucleic acid, or specified portion thereof.

In certain embodiments, compounds described herein also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of a compound. In certain embodiments, the compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 15 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 16 nucleobase portion of a target segment. Also contemplated are compounds that are complementary to at least a 9, 10, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.

Identity

The compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific ION number, or portion thereof. In certain embodiments, compounds described herein are antisense compounds or oligomeric compounds. In certain embodiments, compounds described herein are modified oligonucleotides. As used herein, a compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the compounds described herein as well as compounds having non-identical bases relative to the compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the compound. Percent identity of an compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.

In certain embodiments, compounds described herein, or portions thereof, are, or are at least, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the compounds or SEQ ID NOs, or a portion thereof, disclosed herein. In certain embodiments, compounds described herein are about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or any percentage between such values, to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific ION number, or portion thereof, in which the compounds comprise an oligonucleotide having one or more mismatched nucleobases. In certain such embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 5′-end of the oligonucleotide. In certain such embodiments, the mismatch is at position, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 3′-end of the oligonucleotide.

In certain embodiments, compounds described herein comprise or consist of antisense compounds. In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

In certain embodiments, compounds described herein comprise or consist of oligonucleotides. In certain embodiments, a portion of the oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

Certain Modified Compounds

In certain embodiments, compounds described herein comprise or consist of oligonucleotides consisting of linked nucleosides. Oligonucleotides may be unmodified oligonucleotides (RNA or DNA) or may be modified oligonucleotides. Modified oligonucleotides comprise at least one modification relative to unmodified RNA or DNA (i.e., comprise at least one modified nucleoside (comprising a modified sugar moiety and/or a modified nucleobase) and/or at least one modified internucleoside linkage).

A. Modified Nucleosides

Modified nucleosides comprise a modified sugar moiety or a modified nucleobase or both a modified sugar moiety and a modified nucleobase.

-   -   1. Modified Sugar Moieties

In certain embodiments, sugar moieties are non-bicyclic modified sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties.

In certain embodiments, modified sugar moieties are non-bicyclic modified furanosyl sugar moieties comprising one or more acyclic substituent, including, but not limited, to substituents at the 2′,4′, and/or 5′ positions. In certain embodiments, the furanosyl sugar moiety is a ribosyl sugar moiety. In certain embodiments, one or more acyclic substituent of non-bicyclic modified sugar moieties is branched. Examples of 2′-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or “O-methyl”), and 2′-O(CH₂)₂OCH₃ _(═) (“MOE”). In certain embodiments, 2′-substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, OCN, CF₃, OCF₃, O—C₁-C₁₀ alkoxy, O—C₁-C₁₀ substituted alkoxy, O—C₁-C₁₀ alkyl, O—C₁-C₁₀ substituted alkyl, S-alkyl, N(R_(m))-alkyl, O-alkenyl, S-alkenyl, N(R_(m))-alkenyl, O-alkynyl, S-alkynyl, N(R_(m))-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O(CH₂)₂ON(R_(m))(R_(n)) or OCH₂C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is, independently, H, an amino protecting group, or substituted or unsubstituted C₁-C₁₀ alkyl, and the 2′-substituent groups described in Cook et al., U.S. Pat. No. 6,531,584; Cook et al., U.S. Pat. No. 5,859,221; and Cook et al., U.S. Pat. No. 6,005,087. Certain embodiments of these 2′-substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of 4′-substituent groups suitable for linearly non-bicyclic modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., WO 2015/106128. Examples of 5′-substituent groups suitable for non-bicyclic modified sugar moieties include, but are not limited to: 5′-methyl (R or S), 5′-vinyl, and 5′-methoxy. In certain embodiments, non-bicyclic modified sugars comprise more than one non-bridging sugar substituent, for example, 2′-F-5′-methyl sugar moieties and the modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008/101157 and Rajeev et al., US2013/0203836.

In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclic modified nucleoside comprises a sugar moiety comprising a linear 2′-substituent group selected from: F, NH₂, N₃, OCF₃, OCH₃, O(CH₂)₃NH₂, CH₂CH═CH₂, OCH₂CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O(CH₂)₂ON(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substituted acetamide (OCH₂C(═O)—N(R_(m))(R_(n))), where each R_(m) and R_(n) is, independently, H, an amino protecting group, or substituted or unsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclic modified nucleoside comprises a sugar moiety comprising a linear 2′-substituent group selected from: F, OCF₃, OCH₃, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O(CH₂)₂ON(CH₃)₂, O(CH₂)₂O(CH₂)₂N(CH₃)₂, and OCH₂C(═O)—N(H)CH₃ (“NMA”).

In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclic modified nucleoside comprises a sugar moiety comprising a linear 2′-substituent group selected from: F, OCH₃, and OCH₂CH₂OCH₃.

Nucleosides comprising modified sugar moieties, such as non-bicyclic modified sugar moieties, are referred to by the position(s) of the substitution(s) on the sugar moiety of the nucleoside. For example, nucleosides comprising 2′-substituted or 2′-modified sugar moieties are referred to as 2′-substituted nucleosides or 2′-modified nucleosides.

Certain modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4′ and the 2′ furanose ring atoms. In certain such embodiments, the furanose ring is a ribose ring. Examples of such 4′ to 2′ bridging sugar substituents include, but are not limited to: 4′-CH₂-2′,4′-(CH₂)₂-2′,4′-(CH₂)₃-2′,4′-CH₂—O-2′ (“LNA”), 4′-CH₂—S-2′,4′-(CH₂)₂—O-2′ (“ENA”), 4′-CH(CH₃)—O-2′ (referred to as “constrained ethyl” or “cEt” when in the S configuration), 4′-CH₂—O—CH₂-2′,4′-CH₂—N(R)-2′,4′-CH(CH₂OCH₃)—O-2′ (“constrained MOE” or “cMOE”) and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 7,399,845, Bhat et al., U.S. Pat. No. 7,569,686, Swayze et al., U.S. Pat. No. 7,741,457, and Swayze et al., U.S. Pat. No. 8,022,193), 4′-C(CH₃)(CH₃)—O-2′ and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 8,278,283), 4′-CH₂—N(OCH₃)-2′ and analogs thereof (see, e.g., Prakash et al., U.S. Pat. No. 8,278,425), 4′-CH₂—O—N(CH₃)-2′ (see, e.g., Allerson et al., U.S. Pat. No. 7,696,345 and Allerson et al., U.S. Pat. No. 8,124,745), 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Zhou, et al., J. Org. Chem., 2009, 74, 118-134), 4′-CH₂—C(═CH₂)-2′ and analogs thereof (see e.g., Seth et al., U.S. Pat. No. 8,278,426), 4′-C(R_(a)R_(b))—N(R)—O-2′,4′-C(R_(a)R_(b))—O—N(R)-2′,4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′, wherein each R, R_(a), and R_(b) is, independently, H, a protecting group, or C₁-C₁₂ alkyl (see, e.g. Imanishi et al., U.S. Pat. No. 7,427,672).

In certain embodiments, such 4′ to 2′ bridges independently comprise from 1 to 4 linked groups independently selected from: —[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a))═C(R_(b)), —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—;

-   -   wherein:     -   x is 0, 1, or 2;     -   n is 1, 2, 3, or 4;     -   each R_(a) and R_(b) is, independently, H, a protecting group,         hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂         alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted         C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl,         heterocycle radical, substituted heterocycle radical,         heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,         substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁,         N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl         (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and each J₁ and J₂ is,         independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂         alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted         C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl         (C(═O)—H), substituted acyl, a heterocycle radical, a         substituted heterocycle radical, C₁-C₁₂ aminoalkyl, substituted         C₁-C₁₂ aminoalkyl, or a protecting group.

Additional bicyclic sugar moieties are known in the art, see, for example: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443, Albaek et al., J. Org. Chem., 2006, 71, 7731-7740, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J Am. Chem. Soc., 2007, 129, 8362-8379; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; Wengel et al., U.S. Pat. No. 7,053,207, Imanishi et al., U.S. Pat. No. 6,268,490, Imanishi et al. U.S. Pat. No. 6,770,748, Imanishi et al., U.S. RE44,779; Wengel et al., U.S. Pat. No. 6,794,499, Wengel et al., U.S. Pat. No. 6,670,461; Wengel et al., U.S. Pat. No. 7,034,133, Wengel et al., U.S. Pat. No. 8,080,644; Wengel et al., U.S. Pat. No. 8,034,909; Wengel et al., U.S. Pat. No. 8,153,365; Wengel et al., U.S. Pat. No. 7,572,582; and Ramasamy et al., U.S. Pat. No. 6,525,191, Torsten et al., WO 2004/106356, Wengel et al., WO 1999/014226; Seth et al., WO 2007/134181; Seth et al., U.S. Pat. No. 7,547,684; Seth et al., U.S. Pat. No. 7,666,854; Seth et al., U.S. Pat. No. 8,088,746; Seth et al., U.S. Pat. No. 7,750,131; Seth et al., U.S. Pat. No. 8,030,467; Seth et al., U.S. Pat. No. 8,268,980; Seth et al., U.S. Pat. No. 8,546,556; Seth et al., U.S. Pat. No. 8,530,640; Migawa et al., U.S. Pat. No. 9,012,421; Seth et al., U.S. Pat. No. 8,501,805; Allerson et al., US2008/0039618; and Migawa et al., US2015/0191727.

In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, an LNA nucleoside (described herein) may be in the α-L configuration or in the β-D configuration.

α-L-methyleneoxy (4′-CH₂—O—2′) or α-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). Herein, general descriptions of bicyclic nucleosides include both isomeric configurations. When the positions of specific bicyclic nucleosides (e.g., LNA or cEt) are identified in exemplified embodiments herein, they are in the β-D configuration, unless otherwise specified.

In certain embodiments, modified sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5′-substituted and 4′-2′ bridged sugars).

In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moieties also comprise bridging and/or non-bridging substituents as described herein. For example, certain sugar surrogates comprise a 4′-sulfur atom and a substitution at the 2′-position (see, e.g., Bhat et al., U.S. Pat. No. 7,875,733 and Bhat et al., U.S. Pat. No. 7,939,677) and/or the 5′ position.

In certain embodiments, sugar surrogates comprise rings having other than 5 atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran (“THP”). Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (“HNA”), anitol nucleic acid (“ANA”), manitol nucleic acid (“MNA”) (see e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoro HNA:

(“F-HNA”, see e.g., Swayze et al., U.S. Pat. No. 8,088,904; Swayze et al., U.S. Pat. No. 8,440,803; and Swayze et al., U.S. Pat. No. 9,005,906) F-HNA can also be referred to as a F-THP or 3′-fluoro tetrahydropyran, and nucleosides comprising additional modified THP compounds having the formula:

wherein, independently, for each of said modified THP nucleoside:

-   -   Bx is a nucleobase moiety;     -   T₃ and T₄ are each, independently, an internucleoside linking         group linking the modified THP nucleoside to the remainder of an         oligonucleotide or one of T₃ and T₄ is an internucleoside         linking group linking the modified THP nucleoside to the         remainder of an oligonucleotide and the other of T₃ and T₄ is H,         a hydroxyl protecting group, a linked conjugate group, or a 5′         or 3′-terminal group; q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each,         independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆         alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, or         substituted C₂-C₆ alkynyl; and each of R₁ and R₂ is         independently selected from among: hydrogen, halogen,         substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,         OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁,         and each J₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, modified THP nucleosides are provided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. In certain embodiments, modified THP nucleosides are provided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ is F and R₂ is H, in certain embodiments, R₁ is methoxy and R₂ is H, and in certain embodiments, R₁ is methoxyethoxy and R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. Pat. No. 5,698,685; Summerton et al., U.S. Pat. No. 5,166,315; Summerton et al., U.S. Pat. No. 5,185,444; and Summerton et al., U.S. Pat. No. 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following structure:

In certain embodiments, morpholinos may be modified, for example, by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholinos.”

In certain embodiments, sugar surrogates comprise acyclic moieties. Examples of nucleosides and oligonucleotides comprising such acyclic sugar surrogates include, but are not limited to: peptide nucleic acid (“PNA”), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and nucleosides and oligonucleotides described in Manoharan et al., US2013/130378.

Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used in modified nucleosides.

-   -   2. Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds.

In certain embodiments, compounds described herein comprise modified oligonucleotides. In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising an unmodified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleosides that does not comprise a nucleobase, referred to as an abasic nucleoside.

In certain embodiments, modified nucleobases are selected from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines. In certain embodiments, modified nucleobases are selected from: 2-aminopropyladenine, 5-hydroxymethyl cytosine, 5-methylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (C═C—CH₃) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly, 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in Merigan et al., U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163-166 and 442-443.

Publications that teach the preparation of certain of the above noted modified nucleobases, as well as other modified nucleobases include without limitation, Manoharan et al., US2003/0158403, Manoharan et al., US2003/0175906; Dinh et al., U.S. Pat. No. 4,845,205; Spielvogel et al., U.S. Pat. No. 5,130,302; Rogers et al., U.S. Pat. No. 5,134,066; Bischofberger et al., U.S. Pat. No. 5,175,273; Urdea et al., U.S. Pat. No. 5,367,066; Benner et al., U.S. Pat. No. 5,432,272; Matteucci et al., U.S. Pat. No. 5,434,257; Gmeiner et al., U.S. Pat. No. 5,457,187; Cook et al., U.S. Pat. No. 5,459,255; Froehler et al., U.S. Pat. No. 5,484,908; Matteucci et al., U.S. Pat. No. 5,502,177; Hawkins et al., U.S. Pat. No. 5,525,711; Haralambidis et al., U.S. Pat. No. 5,552,540; Cook et al., U.S. Pat. No. 5,587,469; Froehler et al., U.S. Pat. No. 5,594,121; Switzer et al., U.S. Pat. No. 5,596,091; Cook et al., U.S. Pat. No. 5,614,617; Froehler et al., U.S. Pat. No. 5,645,985; Cook et al., U.S. Pat. No. 5,681,941; Cook et al., U.S. Pat. No. 5,811,534; Cook et al., U.S. Pat. No. 5,750,692; Cook et al., U.S. Pat. No. 5,948,903; Cook et al., U.S. Pat. No. 5,587,470; Cook et al., U.S. Pat. No. 5,457,191; Matteucci et al., U.S. Pat. No. 5,763,588; Froehler et al., U.S. Pat. No. 5,830,653; Cook et al., U.S. Pat. No. 5,808,027; Cook et al., U.S. Pat. No. 6,166,199; and Matteucci et al., U.S. Pat. No. 6,005,096.

In certain embodiments, compounds targeted to a PNPLA3 nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. In certain embodiments, compounds described herein having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

Representative internucleoside linkages having a chiral center include but are not limited to alkylphosphonates and phosphorothioates. Modified oligonucleotides comprising internucleoside linkages having a chiral center can be prepared as populations of modified oligonucleotides comprising stereorandom internucleoside linkages, or as populations of modified oligonucleotides comprising phosphorothioate linkages in particular stereochemical configurations. In certain embodiments, populations of modified oligonucleotides comprise phosphorothioate internucleoside linkages wherein all of the phosphorothioate internucleoside linkages are stereorandom. Such modified oligonucleotides can be generated using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate linkage. Nonetheless, as is well understood by those of skill in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereoconfiguration. In certain embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising one or more particular phosphorothioate internucleoside linkages in a particular, independently selected stereochemical configuration. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 65% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 70% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 80% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 90% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 99% of the molecules in the population. Such chirally enriched populations of modified oligonucleotides can be generated using synthetic methods known in the art, e.g., methods described in Oka et al., JACS 125, 8307 (2003), Wan et al. Nuc. Acid. Res. 42, 13456 (2014), and WO 2017/015555. In certain embodiments, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one indicated phosphorothioate in the (Sp) configuration. In certain embodiments, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one phosphorothioate in the (Rp) configuration. In certain embodiments, modified oligonucleotides comprising (Rp) and/or (Sp) phosphorothioates comprise one or more of the following formulas, respectively, wherein “B” indicates a nucleobase:

Unless otherwise indicated, chiral internucleoside linkages of modified oligonucleotides described herein can be stereorandom or in a particular stereochemical configuration.

In certain embodiments, compounds targeted to a PNPLA3 nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.

In certain embodiments, compounds described herein comprise oligonucleotides. Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.

In certain embodiments, nucleosides of modified oligonucleotides may be linked together using any internucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphates, which contain a phosphodiester bond (“P═O”) (also referred to as unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (“P═S”), and phosphorodithioates (“HS—P═S”). Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂), thiodiester, thionocarbamate (—O—C(═O)(NH)—S—); siloxane (—O—SiH₂—O—); and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—). Modified internucleoside linkages, compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral internucleoside linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.

Neutral internucleoside linkages include, without limitation, phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O—5′), amide-3 (3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal (3′-O—CH₂—O—5′), methoxypropyl, and thioformacetal (3′-S—CH₂—O—5′). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See, for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH₂ component parts.

In certain embodiments, oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, internucleoside linkages are arranged in a gapped motif. In such embodiments, the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region. In certain embodiments, the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate. The nucleoside motif is independently selected, so such oligonucleotides having a gapped internucleoside linkage motif may or may not have a gapped nucleoside motif and, if it does have a gapped nucleoside motif, the wing and gap lengths may or may not be the same.

In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.

In certain embodiments, oligonucleotides comprise one or more methylphosphonate linkages. In certain embodiments, oligonucleotides having a gapmer nucleoside motif comprise a linkage motif comprising all phosphorothioate linkages except for one or two methylphosphonate linkages. In certain embodiments, one methylphosphonate linkage is in the central gap of an oligonucleotide having a gapmer nucleoside motif.

In certain embodiments, it is desirable to arrange the number of phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, it is desirable to arrange the number and position of phosphorothioate internucleoside linkages and the number and position of phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, the number of phosphorothioate internucleoside linkages may be decreased and the number of phosphodiester internucleoside linkages may be increased. In certain embodiments, the number of phosphorothioate internucleoside linkages may be decreased and the number of phosphodiester internucleoside linkages may be increased while still maintaining nuclease resistance. In certain embodiments, it is desirable to decrease the number of phosphorothioate internucleoside linkages while retaining nuclease resistance. In certain embodiments, it is desirable to increase the number of phosphodiester internucleoside linkages while retaining nuclease resistance.

-   -   3. Certain Motifs

In certain embodiments, compounds described herein comprise oligonucleotides. Oligonucleotides can have a motif, e.g. a pattern of unmodified and/or modified sugar moieties, nucleobases, and/or internucleoside linkages. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified sugar. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more modified internucleoside linkage. In such embodiments, the modified, unmodified, and differently modified sugar moieties, nucleobases, and/or internucleoside linkages of a modified oligonucleotide define a pattern or motif. In certain embodiments, the patterns of sugar moieties, nucleobases, and internucleoside linkages are each independent of one another. Thus, a modified oligonucleotide may be described by its sugar motif, nucleobase motif and/or internucleoside linkage motif (as used herein, nucleobase motif describes the modifications to the nucleobases independent of the sequence of nucleobases).

-   -   a. Certain Sugar Motifs

In certain embodiments, compounds described herein comprise oligonucleotides. In certain embodiments, oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif. In certain instances, such sugar motifs include, but are not limited to, any of the sugar modifications discussed herein.

In certain embodiments, modified oligonucleotides comprise or consist of a region having a gapmer motif, which comprises two external regions or “wings” and a central or internal region or “gap”. The three regions of a gapmer motif (the 5′-wing, the gap, and the 3′-wing) form a contiguous sequence of nucleosides, wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3′-most nucleoside of the 5′-wing and the 5′-most nucleoside of the 3′-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap (i.e., the wing/gap junction). In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleosides having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar motif of the 5′-wing differs from the sugar motif of the 3′-wing (asymmetric gapmer).

In certain embodiments, the wings of a gapmer comprise 1-5 nucleosides. In certain embodiments, the wings of a gapmer comprise 2-5 nucleosides. In certain embodiments, the wings of a gapmer comprise 3-5 nucleosides. In certain embodiments, the nucleosides of a gapmer are all modified nucleosides.

In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides. In certain embodiments, the gap of a gapmer comprises 7-10 nucleosides. In certain embodiments, the gap of a gapmer comprises 8-10 nucleosides. In certain embodiments, the gap of a gapmer comprises 10 nucleosides. In certain embodiments, each nucleoside of the gap of a gapmer is an unmodified 2′-deoxy nucleoside.

In certain embodiments, the gapmer is a deoxy gapmer. In such embodiments, the nucleosides on the gap side of each wing/gap junction are unmodified 2′-deoxy nucleosides and the nucleosides on the wing sides of each wing/gap junction are modified nucleosides. In certain such embodiments, each nucleoside of the gap is an unmodified 2′-deoxy nucleoside. In certain such embodiments, each nucleoside of each wing is a modified nucleoside.

In certain embodiments, a modified oligonucleotide has a fully modified sugar motif wherein each nucleoside of the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif wherein each nucleoside of the region comprises a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, a fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of a uniformly modified comprises the same 2′-modification.

-   -   b. Certain Nucleobase Motifs

In certain embodiments, compounds described herein comprise oligonucleotides. In certain embodiments, oligonucleotides comprise modified and/or unmodified nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases are modified. In certain embodiments, each purine or each pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments, some or all of the cytosine nucleobases in a modified oligonucleotide are 5-methylcytosines.

In certain embodiments, modified oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3′-end of the oligonucleotide. In certain embodiments, the block is within 3 nucleosides of the 3′-end of the oligonucleotide. In certain embodiments, the block is at the 5′-end of the oligonucleotide. In certain embodiments, the block is within 3 nucleosides of the 5′-end of the oligonucleotide.

In certain embodiments, oligonucleotides having a gapmer motif comprise a nucleoside comprising a modified nucleobase. In certain such embodiments, one nucleoside comprising a modified nucleobase is in the central gap of an oligonucleotide having a gapmer motif. In certain such embodiments, the sugar moiety of said nucleoside is a 2′-deoxyribosyl moiety. In certain embodiments, the modified nucleobase is selected from: a 2-thiopyrimidine and a 5-propynepyrimidine.

-   -   c. Certain Internucleoside Linkage Motifs

In certain embodiments, compounds described herein comprise oligonucleotides. In certain embodiments, oligonucleotides comprise modified and/or unmodified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, essentially each internucleoside linking group is a phosphate internucleoside linkage (P═O). In certain embodiments, each internucleoside linking group of a modified oligonucleotide is a phosphorothioate (P═S). In certain embodiments, each internucleoside linking group of a modified oligonucleotide is independently selected from a phosphorothioate and phosphate internucleoside linkage. In certain embodiments, the sugar motif of a modified oligonucleotide is a gapmer and the internucleoside linkages within the gap are all modified. In certain such embodiments, some or all of the internucleoside linkages in the wings are unmodified phosphate linkages. In certain embodiments, the terminal internucleoside linkages are modified. In certain embodiments, the sugar motif of a modified oligonucleotide is a gapmer, and the internucleoside linkage motif comprises at least one phosphodiester internucleoside linkage in at least one wing, wherein the at least one phosphodiester linkage is not a terminal internucleoside linkage, and the remaining internucleoside linkages are phosphorothioate internucleoside linkages. In certain such embodiments, all of the phosphorothioate linkages are stereorandom. In certain embodiments, all of the phosphorothioate linkages in the wings are (Sp) phosphorothioates, and the gap comprises at least one Sp, Sp, Rp motif. In certain embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising such internucleoside linkage motifs.

-   -   4. Certain Modified Oligonucleotides

In certain embodiments, compounds described herein comprise modified oligonucleotides. In certain embodiments, the above modifications (sugar, nucleobase, internucleoside linkage) are incorporated into a modified oligonucleotide. In certain embodiments, modified oligonucleotides are characterized by their modification, motifs, and overall lengths. In certain embodiments, such parameters are each independent of one another. Thus, unless otherwise indicated, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. For example, the internucleoside linkages within the wing regions of a sugar gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region of the sugar motif. Likewise, such gapmer oligonucleotides may comprise one or more modified nucleobases independent of the gapmer pattern of the sugar modifications. Furthermore, in certain instances, an oligonucleotide is described by an overall length or range and by lengths or length ranges of two or more regions (e.g., a regions of nucleosides having specified sugar modifications). In such circumstances, it may be possible to select numbers for each range that result in an oligonucleotide having an overall length falling outside the specified range. In such circumstances, both elements must be satisfied. For example, in certain embodiments, a modified oligonucleotide consists of 15-20 linked nucleosides and has a sugar motif consisting of three regions, A, B, and C, wherein region A consists of 2-6 linked nucleosides having a specified sugar motif, region B consists of 6-10 linked nucleosides having a specified sugar motif, and region C consists of 2-6 linked nucleosides having a specified sugar motif. Such embodiments do not include modified oligonucleotides where A and C each consist of 6 linked nucleosides and B consists of 10 linked nucleosides (even though those numbers of nucleosides are permitted within the requirements for A, B, and C) because the overall length of such oligonucleotide will be 22, which exceeds the upper limit of the overall length of the modified oligonucleotide (20). Herein, if a description of an oligonucleotide is silent with respect to one or more parameters, such parameter is not limited. Thus, a modified oligonucleotide described only as having a gapmer sugar motif without further description may have any length, internucleoside linkage motif, and nucleobase motif. Unless otherwise indicated, all modifications are independent of nucleobase sequence.

Certain Conjugated Compounds

In certain embodiments, the compounds described herein comprise or consist of an oligonucleotide (modified or unmodified) and, optionally, one or more conjugate groups and/or terminal groups. Conjugate groups consist of one or more conjugate moiety and a conjugate linker which links the conjugate moiety to the oligonucleotide. Conjugate groups may be attached to either or both ends of an oligonucleotide and/or at any internal position. In certain embodiments, conjugate groups are attached to the 2′-position of a nucleoside of a modified oligonucleotide. In certain embodiments, conjugate groups that are attached to either or both ends of an oligonucleotide are terminal groups. In certain such embodiments, conjugate groups or terminal groups are attached at the 3′ and/or 5′-end of oligonucleotides. In certain such embodiments, conjugate groups (or terminal groups) are attached at the 3′-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 3′-end of oligonucleotides. In certain embodiments, conjugate groups (or terminal groups) are attached at the 5′-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 5′-end of oligonucleotides.

In certain embodiments, the oligonucleotide is modified. In certain embodiments, the oligonucleotide of a compound has a nucleobase sequence that is complementary to a target nucleic acid. In certain embodiments, oligonucleotides are complementary to a messenger RNA (mRNA). In certain embodiments, oligonucleotides are complementary to a pre-mRNA. In certain embodiments, oligonucleotides are complementary to a sense transcript.

Examples of terminal groups include but are not limited to conjugate groups, capping groups, phosphate moieties, protecting groups, modified or unmodified nucleosides, and two or more nucleosides that are independently modified or unmodified.

A. Certain Conjugate Groups

In certain embodiments, oligonucleotides are covalently attached to one or more conjugate groups. In certain embodiments, conjugate groups modify one or more properties of the attached oligonucleotide, including, but not limited to, pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance. In certain embodiments, conjugate groups impart a new property on the attached oligonucleotide, e.g., fluorophores or reporter groups that enable detection of the oligonucleotide.

Certain conjugate groups and conjugate moieties have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic, a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, i, 923-937), a tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; doi: 10.1038/mtna.2014.72 and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or a GalNAc cluster (e.g., WO2014/179620).

-   -   1. Conjugate Moieties

Conjugate moieties include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates (e.g., GalNAc), vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.

In certain embodiments, a conjugate moiety comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial, or an antibiotic.

-   -   2. Conjugate Linkers

Conjugate moieties are attached to oligonucleotides through conjugate linkers. In certain embodiments, a conjugate group is a single chemical bond (i.e. conjugate moiety is attached to an oligonucleotide via a conjugate linker through a single bond). In certain embodiments, the conjugate linker comprises a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units, such as ethylene glycol, nucleosides, or amino acid units.

In certain embodiments, a conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises groups selected from alkyl, amino, oxo, amide and ether groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker includes at least one neutral linking group.

In certain embodiments, conjugate linkers, including the conjugate linkers described above, are bifunctional linking moieties, e.g., those known in the art to be useful for attaching conjugate groups to parent compounds, such as the oligonucleotides provided herein. In general, a bifunctional linking moiety comprises at least two functional groups. One of the functional groups is selected to bind to a particular site on a compound and the other is selected to bind to a conjugate group. Examples of functional groups used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, bifunctional linking moieties comprise one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.

Examples of conjugate linkers include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include, but are not limited to, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl, or substituted or unsubstituted C₂-C₁₀ alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

In certain embodiments, conjugate linkers comprise 1-10 linker-nucleosides. In certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments, such linker-nucleosides comprise a modified sugar moiety. In certain embodiments, linker-nucleosides are unmodified. In certain embodiments, linker-nucleosides comprise an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, a cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is typically desirable for linker-nucleosides to be cleaved from the compound after it reaches a target tissue. Accordingly, linker-nucleosides are typically linked to one another and to the remainder of the compound through cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.

Herein, linker-nucleosides are not considered to be part of the oligonucleotide. Accordingly, in embodiments in which a compound comprises an oligonucleotide consisting of a specified number or range of linked nucleosides and/or a specified percent complementarity to a reference nucleic acid and the compound also comprises a conjugate group comprising a conjugate linker comprising linker-nucleosides, those linker-nucleosides are not counted toward the length of the oligonucleotide and are not used in determining the percent complementarity of the oligonucleotide for the reference nucleic acid. For example, a compound may comprise (1) a modified oligonucleotide consisting of 8-30 nucleosides and (2) a conjugate group comprising 1-10 linker-nucleosides that are contiguous with the nucleosides of the modified oligonucleotide. The total number of contiguous linked nucleosides in such a compound is more than 30. Alternatively, a compound may comprise a modified oligonucleotide consisting of 8-30 nucleosides and no conjugate group. The total number of contiguous linked nucleosides in such a compound is no more than 30. Unless otherwise indicated, conjugate linkers comprise no more than 10 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 5 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 2 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 1 linker-nucleoside.

In certain embodiments, it is desirable for a conjugate group to be cleaved from the oligonucleotide. For example, in certain circumstances, compounds comprising a particular conjugate moiety are better taken up by a particular cell type, but once the compound has been taken up, it is desirable that the conjugate group be cleaved to release the unconjugated or parent oligonucleotide. Thus, certain conjugates may comprise one or more cleavable moieties, typically within the conjugate linker. In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety is a group of atoms comprising at least one cleavable bond. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, a cleavable moiety is selectively cleaved inside a cell or subcellular compartment, such as a lysosome. In certain embodiments, a cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases.

In certain embodiments, a cleavable bond is selected from among: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In certain embodiments, a cleavable bond is one or both of the esters of a phosphodiester. In certain embodiments, a cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a phosphate linkage between an oligonucleotide and a conjugate moiety or conjugate group.

In certain embodiments, a cleavable moiety comprises or consists of one or more linker-nucleosides. In certain such embodiments, one or more linker-nucleosides are linked to one another and/or to the remainder of the compound through cleavable bonds. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, a cleavable moiety is 2′-deoxy nucleoside that is attached to either the 3′ or 5′-terminal nucleoside of an oligonucleotide by a phosphate internucleoside linkage and covalently attached to the remainder of the conjugate linker or conjugate moiety by a phosphate or phosphorothioate linkage. In certain such embodiments, the cleavable moiety is 2′-deoxyadenosine.

-   -   3. Certain Cell-Targeting Conjugate Moieties

In certain embodiments, a conjugate group comprises a cell-targeting conjugate moiety. In certain embodiments, a conjugate group has the general formula:

-   -   wherein n is from 1 to about 3, m is 0 when n is 1, m is 1 when         n is 2 or greater, j is 1 or 0, and k is 1 or 0.

In certain embodiments, n is 1, j is 1 and k is 0. In certain embodiments, n is 1, j is 0 and k is 1. In certain embodiments, n is 1, j is 1 and k is 1. In certain embodiments, n is 2, j is 1 and k is 0. In certain embodiments, n is 2, j is 0 and k is 1. In certain embodiments, n is 2, j is 1 and k is 1. In certain embodiments, n is 3, j is 1 and k is 0. In certain embodiments, n is 3, j is 0 and k is 1. In certain embodiments, n is 3, j is 1 and k is 1.

In certain embodiments, conjugate groups comprise cell-targeting moieties that have at least one tethered ligand. In certain embodiments, cell-targeting moieties comprise two tethered ligands covalently attached to a branching group. In certain embodiments, cell-targeting moieties comprise three tethered ligands covalently attached to a branching group.

In certain embodiments, the cell-targeting moiety comprises a branching group comprising one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino groups. In certain embodiments, the branching group comprises a branched aliphatic group comprising groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino groups. In certain such embodiments, the branched aliphatic group comprises groups selected from alkyl, amino, oxo, amide, and ether groups. In certain such embodiments, the branched aliphatic group comprises groups selected from alkyl, amino, and ether groups. In certain such embodiments, the branched aliphatic group comprises groups selected from alkyl and ether groups. In certain embodiments, the branching group comprises a mono or polycyclic ring system.

In certain embodiments, each tether of a cell-targeting moiety comprises one or more groups selected from alkyl, substituted alkyl, ether, thioether, disulfide, amino, oxo, amide, phosphodiester, and polyethylene glycol, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, thioether, disulfide, amino, oxo, amide, and polyethylene glycol, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, phosphodiester, ether, amino, oxo, and amide, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, amino, oxo, and amide, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, amino, and oxo, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and oxo, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and phosphodiester, in any combination. In certain embodiments, each tether comprises at least one phosphorus linking group or neutral linking group. In certain embodiments, each tether comprises a chain from about 6 to about 20 atoms in length. In certain embodiments, each tether comprises a chain from about 10 to about 18 atoms in length. In certain embodiments, each tether comprises about 10 atoms in chain length.

In certain embodiments, each ligand of a cell-targeting moiety has an affinity for at least one type of receptor on a target cell. In certain embodiments, each ligand has an affinity for at least one type of receptor on the surface of a mammalian liver cell. In certain embodiments, each ligand has an affinity for the hepatic asialoglycoprotein receptor (ASGP-R). In certain embodiments, each ligand is a carbohydrate. In certain embodiments, each ligand is, independently selected from galactose, N-acetyl galactoseamine (GalNAc), mannose, glucose, glucoseamine, and fucose. In certain embodiments, each ligand is N-acetyl galactoseamine (GalNAc). In certain embodiments, the cell-targeting moiety comprises 3 GalNAc ligands. In certain embodiments, the cell-targeting moiety comprises 2 GalNAc ligands. In certain embodiments, the cell-targeting moiety comprises 1 GalNAc ligand.

In certain embodiments, each ligand of a cell-targeting moiety is a carbohydrate, carbohydrate derivative, modified carbohydrate, polysaccharide, modified polysaccharide, or polysaccharide derivative. In certain such embodiments, the conjugate group comprises a carbohydrate cluster (see, e.g., Maier et al., “Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting,” Bioconjugate Chemistry, 2003, 14, 18-29, or Rensen et al., “Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor,” J. Med. Chem. 2004, 47, 5798-5808, which are incorporated herein by reference in their entirety). In certain such embodiments, each ligand is an amino sugar or a thio sugar. For example, amino sugars may be selected from any number of compounds known in the art, such as sialic acid, α-D-galactosamine, β-muramic acid, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, and N-glycoloyl-α-neuraminic acid. For example, thio sugars may be selected from 5-Thio-β-D-glucopyranose, methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio-β-D-galactopyranose, and ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

In certain embodiments, conjugate groups comprise a cell-targeting moiety having the formula:

In certain embodiments, conjugate groups comprise a cell-targeting moiety having the formula:

In certain embodiments, conjugate groups comprise a cell-targeting moiety having the formula:

In certain embodiments, compounds described herein comprise a conjugate group described herein as “LICA-1”. LICA-1 is shown below without the optional cleavable moiety at the end of the conjugate linker:

In certain embodiments, compounds described herein comprise LICA-1 and a cleavable moiety within the conjugate linker have the formula:

-   -   wherein ‘oligo’ is an oligonucleotide.

Representative publications that teach the preparation of certain of the above noted conjugate groups and compounds comprising conjugate groups, tethers, conjugate linkers, branching groups, ligands, cleavable moieties as well as other modifications include, without limitation, U.S. Pat. Nos. 5,994,517, 6,300,319, 6,660,720, 6,906,182, 7,262,177, 7,491,805, 8,106,022, 7,723,509, 9,127,276, US 2006/0148740, US 2011/0123520, WO 2013/033230 and WO 2012/037254, Biessen et al., J. Med. Chem. 1995, 38, 1846-1852, Lee et al., Bioorganic & Medicinal Chemistry 2011, 19, 2494-2500, Rensen et al., J Biol. Chem. 2001, 276, 37577-37584, Rensen et al., J. Med. Chem. 2004, 47, 5798-5808, Sliedregt et al., J. Med. Chem. 1999, 42, 609-618, and Valentijn et al., Tetrahedron, 1997, 53, 759-770, each of which is incorporated by reference herein in its entirety.

In certain embodiments, compounds described herein comprise modified oligonucleotides comprising a gapmer or fully modified motif and a conjugate group comprising at least one, two, or three GalNAc ligands. In certain embodiments, compounds described herein comprise a conjugate group found in any of the following references: Lee, Carbohydr Res, 1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257, 939-945; Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee et al., Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987, 4, 317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676; Biessen et al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al., Tetrahedron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38, 3487-3490; Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato et al., Glycobiol, 2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276, 37577-37584; Lee et al., Methods Enzymol, 2003, 362, 38-43; Westerlind et al., Glycoconj J, 2004, 21, 227-241; Lee et al., BioorgMed Chem Lett, 2006, 16(19), 5132-5135; Maierhofer et al., Bioorg Med Chem, 2007, 15, 7661-7676; Khorev et al., Bioorg Med Chem, 2008, 16, 5216-5231; Lee et al., BioorgMed Chem, 2011, 19, 2494-2500; Kornilova et al., Analyt Biochem, 2012, 425, 43-46; Pujol et al., Angew Chemie Int Ed Engl, 2012, 51, 7445-7448; Biessen et al., J Med Chem, 1995, 38, 1846-1852; Sliedregt et al., J Med Chem, 1999, 42, 609-618; Rensen et al., J Med Chem, 2004, 47, 5798-5808; Rensen et al., Arterioscler Thromb Vasc Biol, 2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464; Sato et al., JAm Chem Soc, 2004, 126, 14013-14022; Lee et al., JOrg Chem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14, 1784-1792; Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et al., Methods Enzymol, 2000, 313, 297-321; Maier et al., Bioconjug Chem, 2003, 14, 18-29; Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan, Antisense Nucleic Acid Drug Dev, 2002, 12, 103-128; Merwin et al., Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013, 21, 5275-5281; International applications WO1998/013381; WO2011/038356; WO1997/046098; WO2008/098788; WO2004/101619; WO2012/037254; WO2011/120053; WO2011/100131; WO2011/163121; WO2012/177947; WO2013/033230; WO2013/075035; WO2012/083185; WO2012/083046; WO2009/082607; WO2009/134487; WO2010/144740; WO2010/148013; WO1997/020563; WO2010/088537; WO2002/043771; WO2010/129709; WO2012/068187; WO2009/126933; WO2004/024757; WO2010/054406; WO2012/089352; WO2012/089602; WO2013/166121; WO2013/165816; U.S. Pat. Nos. 4,751,219; 8,552,163; 6,908,903; 7,262,177; 5,994,517; 6,300,319; 8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812; 6,525,031; 6,660,720; 7,723,509; 8,541,548; 8,344,125; 8,313,772; 8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182; 6,620,916; 8,435,491; 8,404,862; 7,851,615; Published U.S. Patent Application Publications US2011/0097264; US2011/0097265; US2013/0004427; US2005/0164235; US2006/0148740; US2008/0281044; US2010/0240730; US2003/0119724; US2006/0183886; US2008/0206869; US2011/0269814; US2009/0286973; US2011/0207799; US2012/0136042; US2012/0165393; US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075; US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938; US2013/0109817; US2013/0121954; US2013/0178512; US2013/0236968; US2011/0123520; US2003/0077829; US2008/0108801; and US2009/0203132; each of which is incorporated by reference in its entirety.

Compositions and Methods for Formulating Pharmaceutical Compositions

Compounds described herein may be admixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

Certain embodiments provide pharmaceutical compositions comprising one or more compounds or a salt thereof. In certain embodiments, the compounds are antisense compounds or oligomeric compounds. In certain embodiments, the compounds comprise or consist of a modified oligonucleotide. In certain such embodiments, the pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more compound. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution and one or more compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more compound and sterile water. In certain embodiments, a pharmaceutical composition consists of one compound and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises one or more compounds and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more compound and sterile PBS. In certain embodiments, the sterile PBS is pharmaceutical grade PBS. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

A compound described herein targeted to PNPLA3 nucleic acid can be utilized in pharmaceutical compositions by combining the compound with a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutically acceptable diluent is water, such as sterile water suitable for injection. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising a compound targeted to PNPLA3 nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is water. In certain embodiments, the compound comprises or consists of a modified oligonucleotide provided herein.

Pharmaceutical compositions comprising compounds provided herein encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. In certain embodiments, the compounds are antisense compounds or oligomeric compounds. In certain embodiments, the compound comprises or consists of a modified oligonucleotide. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at one or both ends of a compound which are cleaved by endogenous nucleases within the body, to form the active compound.

In certain embodiments, the compounds or compositions further comprise a pharmaceutically acceptable carrier or diluent.

Certain Selected Compounds

Approximately 2,384 newly designed compounds of various lengths, chemistries, and motifs were tested for their effect on human PNPLA3 mRNA in vitro in several cell types (Example 1). Of 2,384 compounds tested for potency at a single dose in vitro, over 400 selected compounds were tested for dose dependent inhibition in A431 cells (Example 2). Of the over 400 compounds tested by dose response assays, the compounds were further screened for high dose tolerability in a BALB/c mouse model and 87 oligonucleotides were selected for in vivo efficacy in a PNPLA3 transgenic mouse model.

Of the 87 oligonucleotides tested in the transgenic mouse model, 23 oligonucleotides were selected to be further tested for tolerability in preclinical rodel models. In the in vivo rodent tolerability models, body weights and organ weights, liver function markers (such as alanine transaminase, aspartate transaminase and bilirubin), and kidney function markers (such as BUN and creatinine) were measured. In the CD1 mouse model and in the Sprague-Dawley rat model, ION 975591, 975605, 975612, 975613, 975616, 975617, 975735, 975736, 994282, and 994284 were found tolerable (Examples 5 and 6).

These compounds were further tested for efficacy in multi-dose assays in PNPLA3 transgenic mice (Example 7).

IONs 994284, 97605, 975616, 994282, 975613, 975617, 975735, 975736, and 975612 were tested for tolerability in cynomolgus monkeys (Example 8). Treatment with the compounds was well tolerated in the monkeys.

Accordingly, provided herein are compounds with any one or more of the improved properties. In certain embodiments, the compounds as described herein are potent and tolerable.

EXAMPLES

The Examples below describe the screening process to identify lead compounds targeted to PNPLA3. ION 994284, 97605, 975616, 994282, 975613, 975617, 975735, 975736, and 975612 resulted in high potency and tolerability.

Non-Limiting Disclosure and Incorporation by Reference

Although the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2′-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2′-OH for the natural 2′-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but not limited to, those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to, such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligonucleotide having the nucleobase sequence “ATCGATCG” encompasses any oligonucleotides having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and compounds having other modified nucleobases, such as “AT^(m)CGAUCG,” wherein ^(m)C indicates a cytosine base comprising a methyl group at the 5-position.

Certain compounds described herein (e.g. modified oligonucleotides) have one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), as a or R, such as for sugar anomers, or as (D) or (L), such as for amino acids, etc. Compounds provided herein that are drawn or described as having certain stereoisomeric configurations include only the indicated compounds. Compounds provided herein that are drawn or described with undefined stereochemistry include all such possible isomers, including their stereorandom and optically pure forms. Likewise, all tautomeric forms of the compounds provided herein are included unless otherwise indicated. Unless otherwise indicated, oligomeric compounds and modified oligonucleotides described herein are intended to include corresponding salt forms.

Compounds described herein include variations in which one or more atoms are replaced with a non-radioactive isotope or radioactive isotope of the indicated element. For example, compounds herein that comprise hydrogen atoms encompass all possible deuterium substitutions for each of the ¹H hydrogen atoms. Isotopic substitutions encompassed by the compounds herein include, but are not limited to: ²H or ³H in place of ¹H, ¹³C or ¹⁴C in place of ¹²C, ¹⁵N in place of ¹⁴N, ¹⁷O or ¹⁸O in place of ¹⁶O, and ³³S, ³⁴S, ³⁵S, or ³⁶S in place of ³²S.

While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.

Example 1: Antisense Inhibition of Human PNPLA3 in A431 Cells

Antisense oligonucleotides were designed targeting a PNPLA3 nucleic acid and were tested for their effects on PNPLA3 mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 3-10-3 cEt gapmers. The gapmers are 16 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three nucleosides. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a cEt sugar modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

“Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human PNPLA3 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_025225.2) or the human PNPLA3 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NC_000022.11 truncated from nucleotides 43921001 to 43954500). ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.

Study 1

Cultured A431 cells at a density of 20,000 cells per well were transfected by free uptake with 4,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PNPLA3 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS36070 (forward sequence CCTTGGTATGTTCCTGCTTCA, designated herein as SEQ ID NO: 11; reverse sequence GTTGTCACTCACTCCTCCATC, designated herein as SEQ ID NO: 12; probe sequence TGGCCTTATCCCTCCTTCCTTCAGA, designated herein as SEQ ID NO: 13) was used to measure mRNA levels. PNPLA3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of PNPLA3, relative to untreated control cells.

TABLE 1 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 912709 27 42 2765 2780 GGCATTCCCAGCGCGA 0 17 912710 95 110 2833 2848 TCCTGATCCGCAGCAG 15 18 912711 103 118 2841 2856 GGCTCGGGTCCTGATC 0 19 912712 131 146 2869 2884 GTTAGGATCTGGGTCG 91 20 912713 164 179 2902 2917 GTACATGGCGGCGGCG 0 21 912714 183 198 2921 2936 TCCAGCCGCGCTCTGC 23 22 912715 196 211 2934 2949 GCGAAGGACAAGCTCC 60 23 912716 197 212 2935 2950 CGCGAAGGACAAGCTC 0 24 912717 272 287 3010 3025 GCGGAGGAGGTGCGGG 0 25 912718 273 288 3011 3026 CGCGGAGGAGGTGCGG 0 26 912719 274 289 3012 3027 TCGCGGAGGAGGTGCG 19 27 912720 290 305 3028 3043 GAACAACATGCGCGCG 0 28 912721 291 306 3029 3044 CGAACAACATGCGCGC 7 29 912722 292 307 3030 3045 CCGAACAACATGCGCG 21 30 912723 293 308 3031 3046 GCCGAACAACATGCGC 0 31 912724 294 309 3032 3047 CGCCGAACAACATGCG 0 32 912725 323 338 3061 3076 GCCGACGCAGTGCAAC 0 33 912726 324 339 3062 3077 CGCCGACGCAGTGCAA 0 34 912727 340 355 3078 3093 GGGATACCGGAGAGGA 43 35 912728 370 385 5944 5959 TCTGAGAGGACCTGCA 53 36 912729 375 390 5949 5964 CAAGATCTGAGAGGAC 64 37 912730 404 419 5978 5993 GCCAATGTTCCGACTC 71 38 912731 410 425 5984 5999 GAAGATGCCAATGTTC 51 39 912732 429 444 6003 6018 TTAAGTTGAAGGATGG 96 40 912733 432 447 6006 6021 TGCTTAAGTTGAAGGA 90 41 912734 478 493 6052 6067 TGGACATTGGCCGGGA 85 42 912735 479 494 6053 6068 GTGGACATTGGCCGGG 50 43 912736 484 499 6058 6073 AGCTGGTGGACATTGG 64 44 912737 528 543 6102 6117 CATCAGACACTCTGGT 5 45 912738 531 546 6105 6120 CCCCATCAGACACTCT 73 46 912739 552 567 6126 6141 AGTCAGACACCAGAAC 54 47 912755 693 708 11911 11926 TGGCATCAATGAAGGG 74 48 912756 698 713 11916 11931 TGTTTTGGCATCAATG 91 49 912757 746 761 11964 11979 TTTAGGGCAGATGTCG 89 50 912758 747 762 11965 11980 CTTTAGGGCAGATGTC 90 51 912759 795 810 12013 12028 GTAGACTGAGCTTGGT 98 52 912760 820 835 12038 12053 AGGTAGAGGTTCCCTG 0 53 912761 841 856 12059 12074 GGGACAAAAGCTCTCG 20 54 912762 873 888 13609 13624 GGCATATCTCTCCCAG 0 55 912763 874 889 13610 13625 AGGCATATCTCTCCCA 0 56 912764 886 901 13622 13637 AAATATCCTCGAAGGC 57 57 912765 888 903 13624 13639 CCAAATATCCTCGAAG 30 58 912766 889 904 13625 13640 TCCAAATATCCTCGAA 38 59 912767 894 909 13630 13645 ATGCATCCAAATATCC 58 60 912768 925 940 N/A N/A TTGCAGATGCCCTTCT 15 61 912769 968 983 16088 16103 ATCCATCCCTTCTGAG 34 62 912770 986 1001 16106 16121 GGGCATGGCGACCTCA 0 63 912771 1004 1019 16124 16139 ACTCATGTTTGCCCAG 67 64 912772 1068 1083 16188 16203 GGTCTAGCAGCTCATC 89 65 912773 1075 1090 16195 16210 CGCAGGTGGTCTAGCA 0 66 912774 1076 1091 16196 16211 ACGCAGGTGGTCTAGC 25 67 912775 1080 1095 16200 16215 TGAGACGCAGGTGGTC 50 68 912776 1086 1101 16206 16221 GGATGCTGAGACGCAG 67 69 912777 1172 1187 19012 19027 GTATCCACCTTTGTCT 78 70 912778 1178 1193 19018 19033 GCTCATGTATCCACCT 79 71 912779 1187 1202 19027 19042 GCAAATCTTGCTCATG 3 72 912780 1188 1203 19028 19043 TGCAAATCTTGCTCAT 13 73 912781 1189 1204 19029 19044 TTGCAAATCTTGCTCA 0 74 912782 1195 1210 19035 19050 AGCAAGTTGCAAATCT 77 75 912783 1199 1214 19039 19054 GGGTAGCAAGTTGCAA 74 76 912784 1205 1220 19045 19060 CCTAATGGGTAGCAAG 62 77 912785 1206 1221 19046 19061 TCCTAATGGGTAGCAA 79 78

TABLE 2 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 912786 1207 1222 19047 19062 ATCCTAATGGGTAGCA 81 79 912787 1211 1226 19051 19066 CATTATCCTAATGGGT 46 80 912788 1212 1227 19052 19067 ACATTATCCTAATGGG 0 81 912789 1213 1228 19053 19068 GACATTATCCTAATGG 70 82 912790 1220 1235 19060 19075 TACATAAGACATTATC 34 83 912791 1224 1239 19064 19079 GCATTACATAAGACAT 86 84 912792 1245 1260 19085 19100 CCACAGGCAGGGTACA 76 85 912793 1246 1261 19086 19101 TCCACAGGCAGGGTAC 28 86 912794 1253 1268 19093 19108 GGCAGATTCCACAGGC 75 87 912795 1259 1274 19099 19114 CGCAATGGCAGATTCC 92 88 912796 1265 1280 19105 19120 GACAATCGCAATGGCA 64 89 912797 1266 1281 19106 19121 GGACAATCGCAATGGC 75 90 912798 1267 1282 19107 19122 TGGACAATCGCAATGG 73 91 912799 1285 1300 23690 23705 AGCCATGTCACCAGTC 67 92 912800 1289 1304 23694 23709 TGGAAGCCATGTCACC 24 93 912801 1290 1305 23695 23710 CTGGAAGCCATGTCAC 72 94 912802 1297 1312 23702 23717 GGCATATCTGGAAGCC 0 95 912803 1298 1313 23703 23718 GGGCATATCTGGAAGC 0 96 912804 1351 1366 23756 23771 AGCACTCGAGTGAACA 0 97 912805 1386 1401 N/A N/A GCATTTGGGACCTGGA 77 98 912806 1387 1402 N/A N/A GGCATTTGGGACCTGG 60 99 912807 1388 1403 25151 25166 TGGCATTTGGGACCTG 41 100 912808 1394 1409 25157 25172 GCTCACTGGCATTTGG 44 101 912809 1523 1538 25286 25301 GTTCAGGCTGGACCTG 49 102 912810 1547 1562 25310 25325 AGGTACTTTATTGCCC 11 103 912811 1550 1565 25313 25328 AGCAGGTACTTTATTG 64 104 912812 1653 1668 25416 25431 AACTTTAGCACCTCTG 91 105 912813 1655 1670 25418 25433 GAAACTTTAGCACCTC 88 106 912814 1656 1671 25419 25434 GGAAACTTTAGCACCT 53 107 912815 1669 1684 25432 25447 CTGCACAAAGATGGGA 80 108 912816 1671 1686 25434 25449 AGCTGCACAAAGATGG 45 109 912817 1685 1700 25448 25463 AGCAATGCGGAGGTAG 15 110 912818 1740 1755 25503 25518 ACCAACTCAGCTCAGA 85 111 912819 1741 1756 25504 25519 AACCAACTCAGCTCAG 79 112 912820 1757 1772 25520 25535 TCCTAGCTTTTCATAA 23 113 912821 1788 1803 25551 25566 TGCTGGACCGCTGCAC 0 114 912822 1796 1811 25559 25574 GAGTTAAGTGCTGGAC 93 115 912823 1802 1817 25565 25580 GTATTAGAGTTAAGTG 92 116 912824 1803 1818 25566 25581 TGTATTAGAGTTAAGT 79 117 912825 1806 1821 25569 25584 TGATGTATTAGAGTTA 92 118 912826 1808 1823 25571 25586 GCTGATGTATTAGAGT 80 119 912827 1821 1836 25584 25599 TGAATTAACGCATGCT 83 120 912828 1822 1837 25585 25600 CTGAATTAACGCATGC 78 121 912829 1870 1885 25633 25648 AGTAAGGGACCCTCTG 17 122 912830 1871 1886 25634 25649 CAGTAAGGGACCCTCT 28 123 912831 1872 1887 25635 25650 TCAGTAAGGGACCCTC 77 124 912832 1874 1889 25637 25652 AGTCAGTAAGGGACCC 51 125 912833 1893 1908 25656 25671 ATTAATAGGGCCACGA 80 126 912834 1895 1910 25658 25673 CCATTAATAGGGCCAC 90 127 912835 1896 1911 25659 25674 ACCATTAATAGGGCCA 81 128 912836 1906 1921 25669 25684 GAACAGTCTGACCATT 82 129 912837 1908 1923 25671 25686 TGGAACAGTCTGACCA 31 130 912838 1909 1924 25672 25687 CTGGAACAGTCTGACC 83 131 912839 1911 1926 25674 25689 TGCTGGAACAGTCTGA 72 132 912840 1916 1931 25679 25694 CCTCATGCTGGAACAG 83 133 912841 1928 1943 25691 25706 TCATTCTAAGAACCTC 96 134 912842 1945 1960 25708 25723 ACCCATCCAAACACCT 16 135 912843 1982 1997 25745 25760 ACACATGGGCCAGCCT 70 136 912844 1989 2004 25752 25767 CAAGATCACACATGGG 70 137 912845 2057 2072 25820 25835 GGGACGAACTGCACCC 0 138 912846 2098 2113 25861 25876 TATCATCTTTGCAGAC 81 139 912847 2116 2131 25879 25894 GTTTTTAGTAGTCAAG 91 140 912848 2117 2132 25880 25895 CGTTTTTAGTAGTCAA 91 141 912849 2145 2160 25908 25923 TATCATCTTGTTACCC 85 142 912850 2148 2163 25911 25926 GATTATCATCTTGTTA 70 143 912851 2150 2165 25913 25928 TAGATTATCATCTTGT 53 144 912852 2151 2166 25914 25929 GTAGATTATCATCTTG 80 145 912853 2152 2167 25915 25930 AGTAGATTATCATCTT 84 146 912854 2175 2190 25938 25953 GTGAAAAAGGTGTTCT 77 147 912855 2182 2197 25945 25960 TAGTTAGGTGAAAAAG 92 148 912856 2188 2203 25951 25966 TTATTTTAGTTAGGTG 88 149 912857 2190 2205 25953 25968 CATTATTTTAGTTAGG 86 150 912858 2273 2288 26036 26051 CTACTAACATCTCACT 55 151 912859 2274 2289 26037 26052 TCTACTAACATCTCAC 89 152 912860 2278 2293 26041 26056 TTATTCTACTAACATC 27 153 912861 2280 2295 26043 26058 GCTTATTCTACTAACA 79 154 912862 2281 2296 26044 26059 GGCTTATTCTACTAAC 81 155 912863 2632 2647 26395 26410 GGTGAATGCCCTGCAC 41 156

TABLE 3 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 912864 2703 2718 26466 26481 TTCAAGTTGTGTGCTC 90 157 912865 2755 2770 26518 26533 GGGAGAAACTCACTGA 37 158 912866 N/A N/A 4416 4431 TGCTACTTGCCCCAGC 2 159 912867 N/A N/A 4421 4436 CACAATGCTACTTGCC 87 160 912868 N/A N/A 4584 4599 CCCAATGGCAGGGCTT 58 161 912869 N/A N/A 4592 4607 TGCTCCTACCCAATGG 46 162 912870 N/A N/A 4766 4781 GACTTTTATTGTTGCT 95 163 912871 N/A N/A 4883 4898 TTCTATACCAGAGTGA 89 164 912872 N/A N/A 4884 4899 TTTCTATACCAGAGTG 89 165 912873 N/A N/A 5405 5420 GTAGATGGCCTTAATG 83 166 912876 N/A N/A 6155 6170 TACATCCACGACTTCG 94 167 912877 N/A N/A 6156 6171 TTACATCCACGACTTC 76 168 912880 N/A N/A 6606 6621 GGAACATTCAGGGTTT 13 169 912881 N/A N/A 6834 6849 ATTACTTGGGTGCAGG 55 170 912884 N/A N/A 6838 6853 GCAGATTACTTGGGTG 45 171 912885 N/A N/A 6931 6946 TGCAGGACAGGTTCCT 30 172 912888 N/A N/A 7549 7564 CACACTGGGTCACCAC 55 173 912889 N/A N/A 7552 7567 AGTCACACTGGGTCAC 61 174 912928 N/A N/A 12273 12288 GGTATATGTTCCCAGG 87 175 912929 N/A N/A 12314 12329 TATAACCACAGCCTGG 29 176 912932 N/A N/A 12321 12336 CTGACTATATAACCAC 81 177 912933 N/A N/A 12666 12681 ATCTTAGTGGCTGGGT 91 178 912936 N/A N/A 12767 12782 CTTACTATGGTAGAGT 88 179 912937 N/A N/A 12768 12783 TCTTACTATGGTAGAG 74 180 912940 N/A N/A 12835 12850 TGCATTGCATAGCCTT 97 181 912941 N/A N/A 12836 12851 TTGCATTGCATAGCCT 96 182 912944 N/A N/A 12907 12922 TGCTTATAAAGCACAC 61 183 912945 N/A N/A 12988 13003 GGAATAAGCCTCCACC 14 184 912948 N/A N/A 14055 14070 GAAATCTGATTGCTTC 59 185 912949 N/A N/A 14393 14408 TACTTATCTGCTCACT 66 186 912952 N/A N/A 14673 14688 TCTCTTAGTGTCCCCA 90 187 14707 14722 912953 N/A N/A 14674 14689 ATCTCTTAGTGTCCCC 92 188 14708 14723 912956 N/A N/A 15284 15299 TCACATTCATGCTTGC 82 189 912957 N/A N/A 15291 15306 GATAACCTCACATTCA 0 190 912960 N/A N/A 15712 15727 GAGCTAGGTGCTTCAC 6 191 912961 N/A N/A 15753 15768 ATAACAACTGAACCAC 85 192 912964 N/A N/A 15937 15952 GTTATTAGCCAAATGC 92 193 912965 N/A N/A 16468 16483 GGAGACTTGGCAAGGT 87 194 912968 N/A N/A 16960 16975 ATTCATGACAGCCCTT 46 195 912969 N/A N/A 17128 17143 ATCGATTTTTCAGAGT 9 196 912972 N/A N/A 17134 17149 ACAAACATCGATTTTT 52 197 912973 N/A N/A 17769 17784 CTCTTTAATGACCTCG 90 198 912976 N/A N/A 18865 18880 GTCAGAGGCACTCACA 25 199 912977 N/A N/A 18959 18974 AGCTATTATCTCCCAC 0 200 912980 N/A N/A 19315 19330 AGTTTCTGGGCTTGCA 90 201 912981 N/A N/A 19382 19397 GGCAATCACAAGAGAC 73 202 912984 N/A N/A 20286 20301 AGAGGAAGCCCAATCA 79 203 20316 20331 912985 N/A N/A 20287 20302 CAGAGGAAGCCCAATC 93 204 20317 20332 912988 N/A N/A 20658 20673 TAGAAATTGCAGTGCC 92 205 912989 N/A N/A 20731 20746 TCCTATCCATATATTG 55 206 912992 N/A N/A 21408 21423 GCAATTCTAGACATGG 88 207 912993 N/A N/A 21558 21573 AGGACTTACACCAAGA 86 208 912996 N/A N/A 21936 21951 TTCCTAATAAGAGCCC 24 209 912997 N/A N/A 21946 21961 GTCAAACATCTTCCTA 66 210 913000 N/A N/A 22077 22092 AAAACTGTAGGATAGG 47 211 913001 N/A N/A 22162 22177 GTTACATCCATAAAAC 0 212 913004 N/A N/A 22169 22184 AGAGAATGTTACATCC 62 213 913008 N/A N/A 23083 23098 AAAGATTAATCAGGGC 61 214 913012 N/A N/A 23788 23803 GTATTTACCTGGAGGC 0 215 913016 N/A N/A 24426 24441 GGCCTATGATTTTCAG 0 216

TABLE 4 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 912874 N/A N/A 5869 5884 ATACTTTTGGCAAGGC 96 217 912875 N/A N/A 5870 5885 AATACTTTTGGCAAGG 91 218 912878 N/A N/A 6159 6174 TGCTTACATCCACGAC 12 219 912879 N/A N/A 6296 6311 CATCATGTTGGTCTCG 54 220 912882 N/A N/A 6835 6850 GATTACTTGGGTGCAG 39 221 912883 N/A N/A 6837 6852 CAGATTACTTGGGTGC 69 222 912886 N/A N/A 7083 7098 TTTAATGGTGTTTTGG 87 223 912887 N/A N/A 7478 7493 TCAAATGCCGGTATTC 52 224 912890 N/A N/A 7587 7602 GTGAACTTCAACTTCC 56 225 912930 N/A N/A 12317 12332 CTATATAACCACAGCC 77 226 912931 N/A N/A 12319 12334 GACTATATAACCACAG 92 227 912934 N/A N/A 12670 12685 AATCATCTTAGTGGCT 91 228 912935 N/A N/A 12765 12780 TACTATGGTAGAGTGG 80 229 912938 N/A N/A 12786 12801 GTACATGGTCTGCAAA 84 230 912939 N/A N/A 12787 12802 TGTACATGGTCTGCAA 57 231 912942 N/A N/A 12843 12858 GCATGCATTGCATTGC 16 232 912943 N/A N/A 12885 12900 ACCAATCCTGTTAGAC 93 233 912946 N/A N/A 13557 13572 GGAGACACCAAGCACC 42 234 912947 N/A N/A 13751 13766 GCACTAAGTGTTAGAA 79 235 912950 N/A N/A 14396 14411 GCTTACTTATCTGCTC 0 236 912951 N/A N/A 14501 14516 GGAGATCCATCCTGCA 0 237 912954 N/A N/A 14675 14690 CATCTCTTAGTGTCCC 92 238 14709 14724 912955 N/A N/A 15122 15137 TCCTAATGTCCTCAAC 9 239 912958 N/A N/A 15293 15308 AAGATAACCTCACATT 33 240 912959 N/A N/A 15294 15309 CAAGATAACCTCACAT 22 241 912962 N/A N/A 15754 15769 TATAACAACTGAACCA 82 242 912963 N/A N/A 15856 15871 GCTTTAAAGCAGGACA 8 243 912966 N/A N/A 16774 16789 AAAATTGTGGGTTTAG 68 244 912967 N/A N/A 16850 16865 ATCATTTGGACCATAG 81 245 912970 N/A N/A 17130 17145 ACATCGATTTTTCAGA 83 246 912971 N/A N/A 17133 17148 CAAACATCGATTTTTC 62 247 912974 N/A N/A 17843 17858 GCTTTACAAGCTGGTC 0 248 912975 N/A N/A 17879 17894 ATCTATGTTCTCCTAG 0 249 912978 N/A N/A 19125 19140 ACCTAAAATGCTCACC 0 250 912979 N/A N/A 19198 19213 CCAGACTACATGCCAC 79 251 912982 N/A N/A 19446 19461 TCTACTAGGCATCTCT 63 252 912983 N/A N/A 19447 19462 TTCTACTAGGCATCTC 42 253 912986 N/A N/A 20288 20303 TCAGAGGAAGCCCAAT 92 254 20318 20333 912987 N/A N/A 20656 20671 GAAATTGCAGTGCCCT 92 255 912990 N/A N/A 21393 21408 GCCAACCTATCACTGA 60 256 912991 N/A N/A 21400 21415 AGACATGGCCAACCTA 32 257 912994 N/A N/A 21565 21580 TGAAATAAGGACTTAC 67 258 912995 N/A N/A 21934 21949 CCTAATAAGAGCCCCA 31 259 912998 N/A N/A 22041 22056 GAAATCTGTCAGAGCA 33 260 912999 N/A N/A 22072 22087 TGTAGGATAGGACTAG 0 261 913002 N/A N/A 22166 22181 GAATGTTACATCCATA 53 262 913003 N/A N/A 22168 22183 GAGAATGTTACATCCA 80 263 913005 N/A N/A 22605 22620 GTGATAAATCTGCAAG 70 264 913006 N/A N/A 23081 23096 AGATTAATCAGGGCCA 8 265 913007 N/A N/A 23082 23097 AAGATTAATCAGGGCC 30 266 913009 N/A N/A 23325 23340 GGTCACATGTGAGCCC 0 267 913010 N/A N/A 23496 23511 CACTTCTGGTTCAAGA 13 268 913011 N/A N/A 23580 23595 CCAATCTGATGACTTC 80 269 913013 N/A N/A 23790 23805 AAGTATTTACCTGGAG 0 270 913014 N/A N/A 24028 24043 CACTCAAAGAGACTCA 65 271 913015 N/A N/A 24425 24440 GCCTATGATTTTCAGG 0 272 913017 N/A N/A 24633 24648 CACTACTGCCCTCTTC 50 273 913018 N/A N/A 24983 24998 TGCTGGGCTGATGTCA 0 274 913019 N/A N/A 25150 25165 GGCATTTGGGACCTGA 67 275

TABLE 5 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915343 1 16 2739 2754 GCCCCCCTCGGACCAT 0 276 915363 45 60 2783 2798 CCTCAGTGTCTCGGCC 0 277 915383 107 122 2845 2860 AATCGGCTCGGGTCCT 29 278 915403 190 205 2928 2943 GACAAGCTCCAGCCGC 64 279 915423 249 264 2987 3002 CGCTCAGGCAGCGGGT 0 280 915443 347 362 N/A N/A CTCCAGCGGGATACCG 6 281 915463 386 401 5960 5975 GGCCTTCCGCACAAGA 0 282 915483 416 431 5990 6005 TGGATGGAAGATGCCA 28 283 915503 452 467 6026 6041 GAGACCCTGTCGGAGG 45 284 915523 488 503 6062 6077 GATGAGCTGGTGGACA 70 285 915543 510 525 6084 6099 GAGAGATGCCTATTTT 92 286 915563 559 574 6133 6148 GACCGAAAGTCAGACA 7 287 915603 697 712 11915 11930 GTTTTGGCATCAATGA 94 288 915623 754 769 11972 11987 GACTTGACTTTAGGGC 98 289 915643 827 842 12045 12060 CGAGAGAAGGTAGAGG 97 290 915663 879 894 13615 13630 CTCGAAGGCATATCTC 66 291 915683 932 947 16052 16067 GGGCCTGTTGCAGATG 0 292 915703 985 1000 16105 16120 GGCATGGCGACCTCAG 6 293 915723 1037 1052 16157 16172 AGCCAAGGCAGCCGAC 0 294 915743 1132 1147 16252 16267 GCGAGCCTGGGCGAGA 0 295 915763 1177 1192 19017 19032 CTCATGTATCCACCTT 88 296 915783 1229 1244 19069 19084 GGGCAGCATTACATAA 73 297 915803 1286 1301 23691 23706 AAGCCATGTCACCAGT 34 298 915823 1348 1363 23753 23768 ACTCGAGTGAACACCT 12 299 915843 1405 1420 25168 25183 GCCTGTTGGCTGCTCA 1 300 915863 1473 1488 25236 25251 CTGCTGGACAGCCCTT 0 301 915883 1542 1557 25305 25320 CTTTATTGCCCAAGAA 72 302 915903 1601 1616 25364 25379 CAGACTCTTCTCTAGT 49 303 915923 1633 1648 25396 25411 AATCTGCTAGACTCGC 88 304 915943 1686 1701 25449 25464 CAGCAATGCGGAGGTA 80 305 915963 1768 1783 25531 25546 GAAAGGTTGCTTCCTA 84 306 915983 1789 1804 25552 25567 GTGCTGGACCGCTGCA 11 307 916003 1815 1830 25578 25593 AACGCATGCTGATGTA 69 308 916023 1848 1863 25611 25626 GCTTCCTGGTGTCATT 81 309 916043 1884 1899 25647 25662 GCCACGAAACAGTCAG 67 310 916063 1913 1928 25676 25691 CATGCTGGAACAGTCT 20 311 916083 1954 1969 25717 25732 AAGGCCCCCACCCATC 0 312 916103 1977 1992 25740 25755 TGGGCCAGCCTACCCC 0 313 916123 2026 2041 25789 25804 GGAAGTGGGATCATGC 55 314 916142 2100 2115 25863 25878 GTTATCATCTTTGCAG 57 315 916162 2139 2154 25902 25917 CTTGTTACCCCCGCCA 84 316 916182 2264 2279 26027 26042 TCTCACTGATTCACAT 83 317 916202 2624 2639 26387 26402 CCCTGCACACTAGATT 55 318 916222 2677 2692 26440 26455 GAGGCGGAAGCTCCTG 0 319 916242 2707 2722 26470 26485 CAGGTTCAAGTTGTGT 83 320 916282 N/A N/A 4225 4240 AAATGTACGGAATCTC 79 321 916302 N/A N/A 4822 4837 GTGTAAACATTTGTCC 74 322 916322 N/A N/A 5414 5429 AGCTTTGGTGTAGATG 49 323 916342 N/A N/A 5801 5816 TACTATGGGAGCCACA 42 324 916362 N/A N/A 6866 6881 TGAAATTGTAACTGCC 70 325 916382 N/A N/A 7492 7507 TAGATCGGTGCTGTTC 27 326 916402 N/A N/A 7785 7800 GTTATAGGCGAGAGCA 0 327 916562 N/A N/A 12316 12331 TATATAACCACAGCCT 58 328 916582 N/A N/A 12932 12947 ATAAGAGCTGTCTCCT 94 329 916602 N/A N/A 13703 13718 CTAGTAAATGCTTGTC 96 330 916622 N/A N/A 14177 14192 CTAATATTTCTACAGC 0 331 916642 N/A N/A 14672 14687 CTCTTAGTGTCCCCAT 95 332 916662 N/A N/A 15542 15557 TTCCATCACAAGGCCT 50 333 916682 N/A N/A 16317 16332 TCCATAATGCACAAGA 71 334 916702 N/A N/A 17223 17238 TGTAGCTGGTTTGTGG 88 335 916722 N/A N/A 18223 18238 AACAGCTACATCAGGC 44 336 916742 N/A N/A 19249 19264 GGCATTGCACATAGAC 74 337 916761 N/A N/A 20410 20425 GTAAGCAATGCAGCCA 88 338 916781 N/A N/A 20659 20674 TTAGAAATTGCAGTGC 91 339 916801 N/A N/A 20989 21004 AGGTATTAAACTGCCA 25 340 916821 N/A N/A 21506 21521 GTCCTAAGAGCACTCA 57 341 916841 N/A N/A 22603 22618 GATAAATCTGCAAGAG 49 342 916861 N/A N/A 23472 23487 GGGACTTACACTGAAA 66 343 916881 N/A N/A 24314 24329 GTCAACGCAGACTGCT 33 344

TABLE 6 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915344 2 17 2740 2755 CGCCCCCCTCGGACCA 0 345 915364 46 61 2784 2799 GCCTCAGTGTCTCGGC 0 346 915384 108 123 2846 2861 GAATCGGCTCGGGTCC 49 347 915404 191 206 2929 2944 GGACAAGCTCCAGCCG 9 348 915424 250 265 2988 3003 TCGCTCAGGCAGCGGG 0 349 915444 348 363 N/A N/A GCTCCAGCGGGATACC 0 350 915464 387 402 5961 5976 TGGCCTTCCGCACAAG 0 351 915484 428 443 6002 6017 TAAGTTGAAGGATGGA 96 352 915504 453 468 6027 6042 AGAGACCCTGTCGGAG 80 353 915524 489 504 6063 6078 AGATGAGCTGGTGGAC 81 354 915544 512 527 6086 6101 AAGAGAGATGCCTATT 77 355 915564 560 575 6134 6149 GGACCGAAAGTCAGAC 0 356 915604 700 715 11918 11933 GTTGTTTTGGCATCAA 91 357 915624 755 770 11973 11988 GGACTTGACTTTAGGG 81 358 915644 828 843 12046 12061 TCGAGAGAAGGTAGAG 24 359 915664 880 895 13616 13631 CCTCGAAGGCATATCT 41 360 915684 952 967 16072 16087 GATGACTTCAGGCCTG 0 361 915704 987 1002 16107 16122 TGGGCATGGCGACCTC 0 362 915724 1038 1053 16158 16173 CAGCCAAGGCAGCCGA 0 363 915744 1133 1148 16253 16268 AGCGAGCCTGGGCGAG 0 364 915764 1179 1194 19019 19034 TGCTCATGTATCCACC 56 365 915784 1230 1245 19070 19085 AGGGCAGCATTACATA 69 366 915804 1293 1308 23698 23713 TATCTGGAAGCCATGT 6 367 915824 1349 1364 23754 23769 CACTCGAGTGAACACC 0 368 915844 1406 1421 25169 25184 GGCCTGTTGGCTGCTC 0 369 915864 1477 1492 25240 25255 GTCTCTGCTGGACAGC 0 370 915884 1545 1560 25308 25323 GTACTTTATTGCCCAA 73 371 915904 1607 1622 25370 25385 GACTCACAGACTCTTC 92 372 915924 1634 1649 25397 25412 GAATCTGCTAGACTCG 65 373 915944 1687 1702 25450 25465 ACAGCAATGCGGAGGT 83 374 915964 1769 1784 25532 25547 CGAAAGGTTGCTTCCT 79 375 915984 1790 1805 25553 25568 AGTGCTGGACCGCTGC 38 376 916004 1816 1831 25579 25594 TAACGCATGCTGATGT 79 377 916024 1849 1864 25612 25627 GGCTTCCTGGTGTCAT 73 378 916044 1885 1900 25648 25663 GGCCACGAAACAGTCA 40 379 916064 1914 1929 25677 25692 TCATGCTGGAACAGTC 80 380 916084 1958 1973 25721 25736 TCACAAGGCCCCCACC 35 381 916104 1978 1993 25741 25756 ATGGGCCAGCCTACCC 0 382 916124 2053 2068 25816 25831 CGAACTGCACCCCTTC 38 383 916143 2101 2116 25864 25879 GGTTATCATCTTTGCA 81 384 916163 2140 2155 25903 25918 TCTTGTTACCCCCGCC 84 385 916183 2265 2280 26028 26043 ATCTCACTGATTCACA 86 386 916203 2625 2640 26388 26403 GCCCTGCACACTAGAT 65 387 916223 2678 2693 26441 26456 GGAGGCGGAAGCTCCT 0 388 916243 2709 2724 26472 26487 GCCAGGTTCAAGTTGT 62 389 916283 N/A N/A 4226 4241 CAAATGTACGGAATCT 52 390 916303 N/A N/A 4864 4879 TACTTTAGGCTCCTGG 90 391 916323 N/A N/A 5422 5437 AGCATTAGAGCTTTGG 75 392 916343 N/A N/A 5803 5818 TCTACTATGGGAGCCA 89 393 916363 N/A N/A 6927 6942 GGACAGGTTCCTTGGA 0 394 916383 N/A N/A 7493 7508 CTAGATCGGTGCTGTT 14 395 916403 N/A N/A 7786 7801 AGTTATAGGCGAGAGC 0 396 916563 N/A N/A 12318 12333 ACTATATAACCACAGC 90 397 916583 N/A N/A 12936 12951 GACAATAAGAGCTGTC 0 398 916603 N/A N/A 13704 13719 GCTAGTAAATGCTTGT 73 399 916623 N/A N/A 14231 14246 CCAACTTTTAGTATTA 92 400 916643 N/A N/A 14678 14693 AGCCATCTCTTAGTGT 50 401 916663 N/A N/A 15566 15581 TCTGATGTCGAAGAGG 68 402 916683 N/A N/A 16341 16356 TCCCATGTGGCAGTAC 0 403 916703 N/A N/A 17239 17254 TCCAAATGCCCAACTC 37 404 916723 N/A N/A 18241 18256 GCAAATAATGTGCACA 22 405 916743 N/A N/A 19250 19265 GGGCATTGCACATAGA 59 406 916762 N/A N/A 20413 20428 GTAGTAAGCAATGCAG 69 407 916782 N/A N/A 20660 20675 CTTAGAAATTGCAGTG 91 408 916802 N/A N/A 21002 21017 ATTTTAACAGCTCAGG 95 409 916822 N/A N/A 21540 21555 TATGACATTTCAGAGT 88 410 916842 N/A N/A 22629 22644 AGTACAAGCGCAGCCT 14 411 916862 N/A N/A 23538 23553 ACAAGGACAAGCCCAC 37 412 916882 N/A N/A 24339 24354 GAAGTAGCGGCATCCC 68 413

TABLE 7 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915345 3 18 2741 2756 CCGCCCCCCTCGGACC 0 414 915365 47 62 2785 2800 TGCCTCAGTGTCTCGG 0 415 915385 109 124 2847 2862 GGAATCGGCTCGGGTC 72 416 915405 193 208 2931 2946 AAGGACAAGCTCCAGC 41 417 915425 251 266 2989 3004 CTCGCTCAGGCAGCGG 0 418 915445 349 364 N/A N/A TGCTCCAGCGGGATAC 0 419 915465 388 403 5962 5977 CTGGCCTTCCGCACAA 16 420 915485 430 445 6004 6019 CTTAAGTTGAAGGATG 27 421 915505 454 469 6028 6043 CAGAGACCCTGTCGGA 72 422 915525 492 507 6066 6081 CGGAGATGAGCTGGTG 92 423 915545 513 528 6087 6102 TAAGAGAGATGCCTAT 57 424 915565 561 576 6135 6150 TGGACCGAAAGTCAGA 0 425 915605 701 716 11919 11934 GGTTGTTTTGGCATCA 97 426 915625 756 771 11974 11989 TGGACTTGACTTTAGG 93 427 915645 829 844 12047 12062 CTCGAGAGAAGGTAGA 0 428 915665 881 896 13617 13632 TCCTCGAAGGCATATC 0 429 915685 953 968 16073 16088 GGATGACTTCAGGCCT 0 430 915705 988 1003 16108 16123 CTGGGCATGGCGACCT 0 431 915725 1039 1054 16159 16174 ACAGCCAAGGCAGCCG 0 432 915745 1134 1149 16254 16269 TAGCGAGCCTGGGCGA 0 433 915765 1193 1208 19033 19048 CAAGTTGCAAATCTTG 0 434 915785 1231 1246 19071 19086 CAGGGCAGCATTACAT 74 435 915805 1300 1315 23705 23720 TCGGGCATATCTGGAA 21 436 915825 1350 1365 23755 23770 GCACTCGAGTGAACAC 0 437 915845 1407 1422 25170 25185 AGGCCTGTTGGCTGCT 0 438 915865 1480 1495 25243 25258 TTGGTCTCTGCTGGAC 21 439 915885 1546 1561 25309 25324 GGTACTTTATTGCCCA 62 440 915905 1609 1624 25372 25387 GTGACTCACAGACTCT 81 441 915925 1635 1650 25398 25413 AGAATCTGCTAGACTC 74 442 915945 1688 1703 25451 25466 CACAGCAATGCGGAGG 56 443 915965 1770 1785 25533 25548 GCGAAAGGTTGCTTCC 66 444 915985 1791 1806 25554 25569 AAGTGCTGGACCGCTG 71 445 916005 1817 1832 25580 25595 TTAACGCATGCTGATG 69 446 916025 1850 1865 25613 25628 GGGCTTCCTGGTGTCA 58 447 916045 1886 1901 25649 25664 GGGCCACGAAACAGTC 9 448 916065 1915 1930 25678 25693 CTCATGCTGGAACAGT 86 449 916085 1959 1974 25722 25737 ATCACAAGGCCCCCAC 82 450 916105 1979 1994 25742 25757 CATGGGCCAGCCTACC 0 451 916125 2054 2069 25817 25832 ACGAACTGCACCCCTT 84 452 916144 2102 2117 25865 25880 AGGTTATCATCTTTGC 90 453 916164 2141 2156 25904 25919 ATCTTGTTACCCCCGC 88 454 916184 2266 2281 26029 26044 CATCTCACTGATTCAC 91 455 916204 2626 2641 26389 26404 TGCCCTGCACACTAGA 47 456 916224 2680 2695 26443 26458 GAGGAGGCGGAAGCTC 0 457 916244 2710 2725 26473 26488 AGCCAGGTTCAAGTTG 71 458 916284 N/A N/A 4227 4242 TCAAATGTACGGAATC 40 459 916304 N/A N/A 4865 4880 GTACTTTAGGCTCCTG 89 460 916324 N/A N/A 5429 5444 ACATATCAGCATTAGA 87 461 916344 N/A N/A 5804 5819 GTCTACTATGGGAGCC 90 462 916364 N/A N/A 6966 6981 GAAGATGCATAGAGGA 0 463 916384 N/A N/A 7550 7565 TCACACTGGGTCACCA 43 464 916544 N/A N/A 12135 12150 GGCAATCAGGGAGGCA 32 465 916564 N/A N/A 12320 12335 TGACTATATAACCACA 92 466 916584 N/A N/A 12951 12966 CCCAATTGCCACTAGG 83 467 916604 N/A N/A 13718 13733 TCTTTACCAAGACCGC 92 468 916624 N/A N/A 14245 14260 GACAAATTCATCAACC 87 469 916644 N/A N/A 14778 14793 CTGTATCCAAAAGGCC 0 470 916664 N/A N/A 15597 15612 ATACATAGCAGAGCCA 44 471 916684 N/A N/A 16352 16367 CACCCTATCGCTCCCA 43 472 916704 N/A N/A 17267 17282 AGTTATGTCTGACTCA 72 473 916724 N/A N/A 18254 18269 AATATACCCCACAGCA 40 474 916744 N/A N/A 19288 19303 GTGCATGTGTGGCTTG 82 475 916763 N/A N/A 20414 20429 TGTAGTAAGCAATGCA 85 476 916783 N/A N/A 20724 20739 CATATATTGCGGATGA 24 477 916803 N/A N/A 21005 21020 GTTATTTTAACAGCTC 95 478 916823 N/A N/A 21561 21576 ATAAGGACTTACACCA 83 479 916843 N/A N/A 22679 22694 CAGCATGCAACCACCC 8 480 916863 N/A N/A 23550 23565 TGGGATGCTAGGACAA 72 481 916883 N/A N/A 24340 24355 GGAAGTAGCGGCATCC 0 482

TABLE 8 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915346 25 40 2763 2778 CATTCCCAGCGCGACG 0 483 915366 52 67 2790 2805 TACCCTGCCTCAGTGT 0 484 915386 112 127 2850 2865 TCGGGAATCGGCTCGG 26 485 915406 195 210 2933 2948 CGAAGGACAAGCTCCA 69 486 915426 252 267 2990 3005 GCTCGCTCAGGCAGCG 0 487 915446 350 365 N/A N/A CTGCTCCAGCGGGATA 0 488 915466 389 404 5963 5978 CCTGGCCTTCCGCACA 40 489 915486 431 446 6005 6020 GCTTAAGTTGAAGGAT 88 490 915506 455 470 6029 6044 GCAGAGACCCTGTCGG 32 491 915526 493 508 6067 6082 CCGGAGATGAGCTGGT 4 492 915546 514 529 6088 6103 GTAAGAGAGATGCCTA 94 493 915566 562 577 6136 6151 TTGGACCGAAAGTCAG 56 494 915606 702 717 11920 11935 TGGTTGTTTTGGCATC 99 495 915626 757 772 11975 11990 GTGGACTTGACTTTAG 89 496 915646 830 845 12048 12063 TCTCGAGAGAAGGTAG 0 497 915666 882 897 13618 13633 ATCCTCGAAGGCATAT 0 498 915686 956 971 16076 16091 TGAGGATGACTTCAGG 10 499 915706 989 1004 16109 16124 GCTGGGCATGGCGACC 10 500 915726 1064 1079 16184 16199 TAGCAGCTCATCTCCC 67 501 915746 1135 1150 16255 16270 GTAGCGAGCCTGGGCG 0 502 915766 1196 1211 19036 19051 TAGCAAGTTGCAAATC 78 503 915786 1232 1247 19072 19087 ACAGGGCAGCATTACA 87 504 915806 1302 1317 23707 23722 CGTCGGGCATATCTGG 53 505 915826 1352 1367 23757 23772 CAGCACTCGAGTGAAC 24 506 915846 1408 1423 25171 25186 GAGGCCTGTTGGCTGC 0 507 915866 1508 1523 25271 25286 GAGGATGGACCGCGGG 0 508 915886 1549 1564 25312 25327 GCAGGTACTTTATTGC 0 509 915906 1610 1625 25373 25388 AGTGACTCACAGACTC 35 510 915926 1636 1651 25399 25414 AAGAATCTGCTAGACT 69 511 915946 1689 1704 25452 25467 ACACAGCAATGCGGAG 69 512 915966 1771 1786 25534 25549 GGCGAAAGGTTGCTTC 58 513 915986 1792 1807 25555 25570 TAAGTGCTGGACCGCT 70 514 916006 1818 1833 25581 25596 ATTAACGCATGCTGAT 73 515 916026 1851 1866 25614 25629 TGGGCTTCCTGGTGTC 71 516 916046 1887 1902 25650 25665 AGGGCCACGAAACAGT 61 517 916066 1917 1932 25680 25695 ACCTCATGCTGGAACA 81 518 916086 1960 1975 25723 25738 CATCACAAGGCCCCCA 48 519 916106 1980 1995 25743 25758 ACATGGGCCAGCCTAC 54 520 916126 2055 2070 25818 25833 GACGAACTGCACCCCT 77 521 916145 2105 2120 25868 25883 TCAAGGTTATCATCTT 89 522 916165 2142 2157 25905 25920 CATCTTGTTACCCCCG 89 523 916185 2270 2285 26033 26048 CTAACATCTCACTGAT 66 524 916205 2627 2642 26390 26405 ATGCCCTGCACACTAG 62 525 916225 2681 2696 26444 26459 AGAGGAGGCGGAAGCT 25 526 916245 2711 2726 26474 26489 AAGCCAGGTTCAAGTT 83 527 916285 N/A N/A 4240 4255 ATTAGGACAAGATTCA 75 528 916305 N/A N/A 4866 4881 TGTACTTTAGGCTCCT 93 529 916325 N/A N/A 5430 5445 AACATATCAGCATTAG 85 530 916345 N/A N/A 5839 5854 CAAGGATGCCACCAAC 84 531 916365 N/A N/A 6974 6989 TCATTATGGAAGATGC 0 532 916385 N/A N/A 7602 7617 TTAACAACCCTGTCAG 1 533 916545 N/A N/A 12151 12166 GTAACTGGTAGCTCCT 93 534 916565 N/A N/A 12338 12353 ACCCATACTGCACCCC 79 535 916585 N/A N/A 12957 12972 GCCTATCCCAATTGCC 70 536 916605 N/A N/A 13719 13734 GTCTTTACCAAGACCG 23 537 916625 N/A N/A 14248 14263 AACGACAAATTCATCA 84 538 916645 N/A N/A 14788 14803 TGCAATCCCCCTGTAT 17 539 916665 N/A N/A 15598 15613 AATACATAGCAGAGCC 68 540 916685 N/A N/A 16366 16381 TGTCATGGTTGCCTCA 70 541 916705 N/A N/A 17273 17288 ATAAGGAGTTATGTCT 80 542 916725 N/A N/A 18255 18270 GAATATACCCCACAGC 58 543 916745 N/A N/A 19295 19310 GTTACAGGTGCATGTG 75 544 916764 N/A N/A 20435 20450 AGTCATCTGGAGTCAC 69 545 916784 N/A N/A 20756 20771 TCAGACAACCCACTGA 24 546 916804 N/A N/A 21046 21061 AGGAATCTGAATCCTA 0 547 916824 N/A N/A 21640 21655 GATAATTTCCTAGAGC 29 548 916844 N/A N/A 22699 22714 GAAATAAGTGCTCAGG 73 549 916864 N/A N/A 23582 23597 CTCCAATCTGATGACT 53 550 916884 N/A N/A 24347 24362 GAATTCAGGAAGTAGC 50 551

TABLE 9 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915347   26   41  2764  2779 GCATTCCCAGCGCGAC  0 552 915367   58   73  2796  2811 GCTCTCTACCCTGCCT  9 553 915387  113  128  2851  2866 ATCGGGAATCGGCTCG 28 554 915407  198  213  2936  2951 CCGCGAAGGACAAGCT  0 555 915427  253  268  2991  3006 TGCTCGCTCAGGCAGC  0 556 915447  351  366 N/A N/A TCTGCTCCAGCGGGAT  1 557 915467  391  406  5965  5980 CTCCTGGCCTTCCGCA 29 558 915487  433  448  6007  6022 TTGCTTAAGTTGAAGG 94 559 915507  456  471  6030  6045 TGCAGAGACCCTGTCG 31 560 915527  494  509  6068  6083 GCCGGAGATGAGCTGG  0 561 915547  515  530  6089  6104 GGTAAGAGAGATGCCT  0 562 915567  563  578  6137  6152 TTTGGACCGAAAGTCA  0 563 915607  703  718 11921 11936 ATGGTTGTTTTGGCAT 35 564 915627  758  773 11976 11991 CGTGGACTTGACTTTA 85 565 915647  831  846 12049 12064 CTCTCGAGAGAAGGTA  7 566 915667  883  898 13619 13634 TATCCTCGAAGGCATA  0 567 915687  959  974 16079 16094 TTCTGAGGATGACTTC 39 568 915707  996 1011 16116 16131 TTGCCCAGCTGGGCAT  0 569 915727 1065 1080 16185 16200 CTAGCAGCTCATCTCC 58 570 915747 1136 1151 16256 16271 TGTAGCGAGCCTGGGC 16 571 915767 1197 1212 19037 19052 GTAGCAAGTTGCAAAT 80 572 915787 1233 1248 19073 19088 TACAGGGCAGCATTAC 71 573 915807 1316 1331 23721 23736 CAACCACAGGACATCG  0 574 915827 1353 1368 23758 23773 TCAGCACTCGAGTGAA  0 575 915847 1409 1424 25172 25187 GGAGGCCTGTTGGCTG  0 576 915867 1509 1524 25272 25287 TGAGGATGGACCGCGG 14 577 915887 1553 1568 25316 25331 ACCAGCAGGTACTTTA 29 578 915907 1611 1626 25374 25389 AAGTGACTCACAGACT 29 579 915927 1637 1652 25400 25415 AAAGAATCTGCTAGAC 60 580 915947 1690 1705 25453 25468 TACACAGCAATGCGGA 69 581 915967 1772 1787 25535 25550 AGGCGAAAGGTTGCTT  0 582 915987 1793 1808 25556 25571 TTAAGTGCTGGACCGC 82 583 916007 1819 1834 25582 25597 AATTAACGCATGCTGA 61 584 916027 1864 1879 25627 25642 GGACCCTCTGCACTGG 43 585 916047 1888 1903 25651 25666 TAGGGCCACGAAACAG 80 586 916067 1918 1933 25681 25696 AACCTCATGCTGGAAC 72 587 916087 1961 1976 25724 25739 CCATCACAAGGCCCCC 63 588 916107 1981 1996 25744 25759 CACATGGGCCAGCCTA 74 589 916127 2056 2071 25819 25834 GGACGAACTGCACCCC  5 590 916146 2106 2121 25869 25884 GTCAAGGTTATCATCT 88 591 916166 2143 2158 25906 25921 TCATCTTGTTACCCCC 90 592 916186 2272 2287 26035 26050 TACTAACATCTCACTG  1 593 916206 2628 2643 26391 26406 AATGCCCTGCACACTA 56 594 916226 2682 2697 26445 26460 GAGAGGAGGCGGAAGC 10 595 916246 2712 2727 26475 26490 TAAGCCAGGTTCAAGT 81 596 916286 N/A N/A  4244  4259 TTTCATTAGGACAAGA 61 597 916306 N/A N/A  4867  4882 GTGTACTTTAGGCTCC 97 598 916326 N/A N/A  5431  5446 GAACATATCAGCATTA 52 599 916346 N/A N/A  5872  5887 GTAATACTTTTGGCAA 75 600 916366 N/A N/A  7069  7084 GGTATTACAAATTATC 10 601 916386 N/A N/A  7603  7618 CTTAACAACCCTGTCA  0 602 916546 N/A N/A 12152 12167 AGTAACTGGTAGCTCC 88 603 916566 N/A N/A 12343 12358 CTAATACCCATACTGC 84 604 916586 N/A N/A 12966 12981 AACTTTGCAGCCTATC 85 605 916606 N/A N/A 13739 13754 AGAACTAAGGCAAATC 85 606 916626 N/A N/A 14257 14272 GTCTTGGCCAACGACA  0 607 916646 N/A N/A 14793 14808 CAGGATGCAATCCCCC 45 608 916666 N/A N/A 15601 15616 GCCAATACATAGCAGA 75 609 916686 N/A N/A 16630 16645 GTCCATGAAATCCAGG  0 610 916706 N/A N/A 17293 17308 TCTCTTAGGGCACCTC 87 611 916726 N/A N/A 18256 18271 TGAATATACCCCACAG 24 612 916746 N/A N/A 19337 19352 AGCTCTAGGAGTCCCC 63 613 916765 N/A N/A 20513 20528 CCAGATTGAGTCTCCT 91 614 916785 N/A N/A 20775 20790 AATCAAGTGCCCTCCA 73 615 916805 N/A N/A 21211 21226 TGTAGCTGTGTGGTGG 85 616 916825 N/A N/A 21760 21775 TACCATGATCAGGTCA  0 617 916845 N/A N/A 22713 22728 GTAAAGATGTGAGTGA 85 618 916865 N/A N/A 23606 23621 GTTTACAAAAGCTGCC 17 619 916885 N/A N/A 24375 24390 TGAACTCCGGCTCAGT  0 620

TABLE 10 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915348   28   43  2766  2781 GGGCATTCCCAGCGCG  0 621 915368   59   74  2797  2812 CGCTCTCTACCCTGCC  0 622 915388  114  129  2852  2867 GATCGGGAATCGGCTC 32 623 915408  199  214  2937  2952 CCCGCGAAGGACAAGC  6 624 915428  275  290  3013  3028 GTCGCGGAGGAGGTGC  0 625 915448  352  367 N/A N/A GTCTGCTCCAGCGGGA  4 626 915468  392  407  5966  5981 ACTCCTGGCCTTCCGC 87 627 915488  434  449  6008  6023 CTTGCTTAAGTTGAAG  0 628 915508  457  472  6031  6046 TTGCAGAGACCCTGTC 63 629 915528  495  510  6069  6084 TGCCGGAGATGAGCTG  0 630 915548  516  531  6090  6105 TGGTAAGAGAGATGCC 18 631 915568  564  579  6138  6153 CTTTGGACCGAAAGTC 10 632 915608  704  719 11922 11937 GATGGTTGTTTTGGCA 98 633 915628  772  787 11990 12005 ACATGAAGAAAGTTCG 41 634 915648  832  847 12050 12065 GCTCTCGAGAGAAGGT 55 635 915668  884  899 13620 13635 ATATCCTCGAAGGCAT 33 636 915688  962  977 16082 16097 CCCTTCTGAGGATGAC 11 637 915708  998 1013 16118 16133 GTTTGCCCAGCTGGGC  0 638 915728 1067 1082 16187 16202 GTCTAGCAGCTCATCT 68 639 915748 1137 1152 16257 16272 CTGTAGCGAGCCTGGG  0 640 915768 1198 1213 19038 19053 GGTAGCAAGTTGCAAA 90 641 915788 1234 1249 19074 19089 GTACAGGGCAGCATTA 69 642 915808 1317 1332 23722 23737 GCAACCACAGGACATC 51 643 915828 1354 1369 23759 23774 ATCAGCACTCGAGTGA  0 644 915848 1410 1425 25173 25188 GGGAGGCCTGTTGGCT 17 645 915868 1510 1525 25273 25288 CTGAGGATGGACCGCG 53 646 915888 1554 1569 25317 25332 CACCAGCAGGTACTTT  0 647 915908 1612 1627 25375 25390 CAAGTGACTCACAGAC 91 648 915928 1639 1654 25402 25417 TGAAAGAATCTGCTAG 59 649 915948 1691 1706 25454 25469 CTACACAGCAATGCGG 20 650 915968 1773 1788 25536 25551 CAGGCGAAAGGTTGCT 60 651 915988 1794 1809 25557 25572 GTTAAGTGCTGGACCG 86 652 916008 1820 1835 25583 25598 GAATTAACGCATGCTG 88 653 916028 1865 1880 25628 25643 GGGACCCTCTGCACTG  0 654 916048 1889 1904 25652 25667 ATAGGGCCACGAAACA 75 655 916068 1919 1934 25682 25697 GAACCTCATGCTGGAA 72 656 916088 1962 1977 25725 25740 CCCATCACAAGGCCCC 37 657 916108 1984 1999 25747 25762 TCACACATGGGCCAGC 84 658 916128 2079 2094 25842 25857 CTGACAGGCAGTGTCG 10 659 916147 2107 2122 25870 25885 AGTCAAGGTTATCATC 81 660 916167 2144 2159 25907 25922 ATCATCTTGTTACCCC 88 661 916187 2276 2291 26039 26054 ATTCTACTAACATCTC 90 662 916207 2629 2644 26392 26407 GAATGCCCTGCACACT 72 663 916227 2691 2706 26454 26469 GCTCCAGTGGAGAGGA 14 664 916247 2713 2728 26476 26491 ATAAGCCAGGTTCAAG 88 665 916287 N/A N/A  4308  4323 GTGAGAAACAAACCCT 92 666 916307 N/A N/A  4882  4897 TCTATACCAGAGTGAG 84 667 916327 N/A N/A  5514  5529 AGGAATGAGTCTCCCA 17 668 916347 N/A N/A  5873  5888 GGTAATACTTTTGGCA 70 669 916367 N/A N/A  7106  7121 CGCTTATGAAAGCATC  0 670 916387 N/A N/A  7605  7620 CCCTTAACAACCCTGT 28 671 916547 N/A N/A 12167 12182 TTTGATTGTGCAGACA 98 672 916567 N/A N/A 12345 12360 TCCTAATACCCATACT 74 673 916587 N/A N/A 12969 12984 ACAAACTTTGCAGCCT 95 674 916607 N/A N/A 13742 13757 GTTAGAACTAAGGCAA 94 675 916627 N/A N/A 14301 14316 GAGCAGATAAATACAC 91 676 916647 N/A N/A 14892 14907 TGGTATCTCGCTTCCT  0 677 916667 N/A N/A 15613 15628 TAAAGCCACGCAGCCA 46 678 916687 N/A N/A 16656 16671 CCAGATGCAGGACCCC  0 679 916707 N/A N/A 17326 17341 AAACTAATGCACCTGG 43 680 916727 N/A N/A 18257 18272 CTGAATATACCCCACA 75 681 916747 N/A N/A 19360 19375 AGCTGCTATGTGAGGC 12 682 916766 N/A N/A 20520 20535 TCAGTAACCAGATTGA 25 683 916786 N/A N/A 20778 20793 TTTAATCAAGTGCCCT 81 684 916806 N/A N/A 21216 21231 CAGGATGTAGCTGTGT 84 685 916826 N/A N/A 21887 21902 TAAGATCCCATCTTAC 13 686 916846 N/A N/A 22739 22754 AAAGTAAACACCCACC 42 687 916866 N/A N/A 23625 23640 GCTTACAACACTACCC 57 688 916886 N/A N/A 24393 24408 GTAATGGGAGCCAGGC 38 689

TABLE 11 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915349   29   44  2767  2782 AGGGCATTCCCAGCGC  0 690 915369   60   75  2798  2813 GCGCTCTCTACCCTGC 23 691 915389  115  130  2853  2868 GGATCGGGAATCGGCT 54 692 915409  200  215  2938  2953 GCCCGCGAAGGACAAG 32 693 915429  276  291  3014  3029 CGTCGCGGAGGAGGTG  0 694 915449  364  379  5938  5953 AGGACCTGCAGAGTCT 21 695 915469  394  409  5968  5983 CGACTCCTGGCCTTCC 59 696 915489  435  450  6009  6024 ACTTGCTTAAGTTGAA 86 697 915509  466  481  6040  6055 GGGAGGCATTTGCAGA 57 698 915529  496  511  6070  6085 TTGCCGGAGATGAGCT 40 699 915549  518  533  6092  6107 TCTGGTAAGAGAGATG 61 700 915569  565  580  6139  6154 TCTTTGGACCGAAAGT  9 701 915609  705  720 11923 11938 TGATGGTTGTTTTGGC 99 702 915629  776  791 11994 12009 GTCCACATGAAGAAAG 32 703 915649  833  848 12051 12066 AGCTCTCGAGAGAAGG 36 704 915669  885  900 13621 13636 AATATCCTCGAAGGCA 55 705 915689  969  984 16089 16104 GATCCATCCCTTCTGA 20 706 915709  999 1014 16119 16134 TGTTTGCCCAGCTGGG  5 707 915729 1077 1092 16197 16212 GACGCAGGTGGTCTAG  0 708 915749 1138 1153 N/A N/A GCTGTAGCGAGCCTGG 71 709 915769 1200 1215 19040 19055 TGGGTAGCAAGTTGCA 81 710 915789 1235 1250 19075 19090 GGTACAGGGCAGCATT 88 711 915809 1318 1333 23723 23738 TGCAACCACAGGACAT 40 712 915829 1355 1370 23760 23775 CATCAGCACTCGAGTG  0 713 915849 1424 1439 25187 25202 CTCAGGTGTGCATGGG 61 714 915869 1511 1526 25274 25289 CCTGAGGATGGACCGC 70 715 915889 1556 1571 25319 25334 AGCACCAGCAGGTACT 35 716 915909 1613 1628 25376 25391 TCAAGTGACTCACAGA 84 717 915929 1645 1660 25408 25423 CACCTCTGAAAGAATC 89 718 915949 1692 1707 25455 25470 ACTACACAGCAATGCG 33 719 915969 1774 1789 25537 25552 ACAGGCGAAAGGTTGC 88 720 915989 1795 1810 25558 25573 AGTTAAGTGCTGGACC 84 721 916009 1823 1838 25586 25601 GCTGAATTAACGCATG 67 722 916029 1866 1881 25629 25644 AGGGACCCTCTGCACT 15 723 916049 1890 1905 25653 25668 AATAGGGCCACGAAAC 52 724 916069 1920 1935 25683 25698 AGAACCTCATGCTGGA 85 725 916089 1963 1978 25726 25741 CCCCATCACAAGGCCC 20 726 916109 1985 2000 25748 25763 ATCACACATGGGCCAG 72 727 916129 2080 2095 25843 25858 CCTGACAGGCAGTGTC 15 728 916148 2108 2123 25871 25886 TAGTCAAGGTTATCAT 87 729 916168 2146 2161 25909 25924 TTATCATCTTGTTACC 82 730 916188 2279 2294 26042 26057 CTTATTCTACTAACAT 87 731 916208 2630 2645 26393 26408 TGAATGCCCTGCACAC 68 732 916228 2692 2707 26455 26470 TGCTCCAGTGGAGAGG 80 733 916248 2726 2741 26489 26504 GTCCCTGCAGAAAATA  0 734 916288 N/A N/A  4337  4352 AGCATACCACACCCCA 75 735 916308 N/A N/A  5086  5101 GGACATGCTCAGCAGC 68 736 916328 N/A N/A  5533  5548 TGCTGTAGGCCTCAGC  0 737 916348 N/A N/A  5874  5889 TGGTAATACTTTTGGC 86 738 916368 N/A N/A  7132  7147 GTAAATGGAGTCCTTC 80 739 916388 N/A N/A  7612  7627 CATAATCCCCTTAACA 32 740 916548 N/A N/A 12195 12210 TTAACCATCAAGGACA 77 741 916568 N/A N/A 12665 12680 TCTTAGTGGCTGGGTA 85 742 916588 N/A N/A 12973 12988 CCTAACAAACTTTGCA 32 743 916608 N/A N/A 13749 13764 ACTAAGTGTTAGAACT 76 744 916628 N/A N/A 14338 14353 CTGCAGTATCCCTAGC  0 745 916648 N/A N/A 15012 15027 TCCCATCGGTCATTTC 45 746 916668 N/A N/A 15682 15697 GAAACCACTATCATCA 62 747 916688 N/A N/A 16671 16686 GTAATAGGCCAAGTCC  0 748 916708 N/A N/A 17327 17342 CAAACTAATGCACCTG 66 749 916728 N/A N/A 18332 18347 CCAATATCATAGCTGA 85 750 916748 N/A N/A 19376 19391 CACAAGAGACTGGACC 64 751 916767 N/A N/A 20551 20566 TACTATGGGATGAGTA  0 752 916787 N/A N/A 20779 20794 TTTTAATCAAGTGCCC 38 753 916807 N/A N/A 21218 21233 GGCAGGATGTAGCTGT 63 754 916827 N/A N/A 21947 21962 AGTCAAACATCTTCCT 50 755 916847 N/A N/A 22759 22774 CAGACTAACTTACTAA 77 756 916867 N/A N/A 23626 23641 AGCTTACAACACTACC 13 757 916887 N/A N/A 24505 24520 ATGCTACGGGCTCTCA  0 758

TABLE 12 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915350   30   45  2768  2783 CAGGGCATTCCCAGCG  0 759 915370   82   97  2820  2835 CAGCTCCGCCCGGCGC 14 760 915390  130  145  2868  2883 TTAGGATCTGGGTCGG 88 761 915410  201  216  2939  2954 AGCCCGCGAAGGACAA  0 762 915430  295  310  3033  3048 GCGCCGAACAACATGC  0 763 915450  366  381  5940  5955 AGAGGACCTGCAGAGT 83 764 915470  395  410  5969  5984 CCGACTCCTGGCCTTC 68 765 915490  436  451  6010  6025 AACTTGCTTAAGTTGA 41 766 915510  467  482  6041  6056 CGGGAGGCATTTGCAG 44 767 915530  497  512  6071  6086 TTTGCCGGAGATGAGC 92 768 915550  519  534  6093  6108 CTCTGGTAAGAGAGAT 20 769 915570  566  581  6140  6155 GTCTTTGGACCGAAAG 19 770 915590  627  642  7857  7872 GAGGGATAAGGCCACT 83 771 915610  706  721 11924 11939 GTGATGGTTGTTTTGG 97 772 915630  782  797 12000 12015 GGTGATGTCCACATGA 87 773 915650  834  849 12052 12067 AAGCTCTCGAGAGAAG 44 774 915670  887  902 13623 13638 CAAATATCCTCGAAGG  0 775 915690  970  985 16090 16105 GGATCCATCCCTTCTG  0 776 915710 1003 1018 16123 16138 CTCATGTTTGCCCAGC 68 777 915730 1078 1093 16198 16213 AGACGCAGGTGGTCTA  0 778 915750 1139 1154 N/A N/A TGCTGTAGCGAGCCTG 56 779 915770 1201 1216 19041 19056 ATGGGTAGCAAGTTGC 79 780 915790 1247 1262 19087 19102 TTCCACAGGCAGGGTA 48 781 915810 1320 1335 23725 23740 ACTGCAACCACAGGAC 22 782 915830 1356 1371 23761 23776 ACATCAGCACTCGAGT  0 783 915850 1427 1442 25190 25205 CTGCTCAGGTGTGCAT 10 784 915870 1512 1527 25275 25290 ACCTGAGGATGGACCG 69 785 915890 1557 1572 25320 25335 CAGCACCAGCAGGTAC 62 786 915910 1617 1632 25380 25395 CTCCTCAAGTGACTCA 83 787 915930 1648 1663 25411 25426 TAGCACCTCTGAAAGA 55 788 915950 1693 1708 25456 25471 CACTACACAGCAATGC 74 789 915970 1775 1790 25538 25553 CACAGGCGAAAGGTTG 72 790 915990 1797 1812 25560 25575 AGAGTTAAGTGCTGGA 92 791 916010 1824 1839 25587 25602 AGCTGAATTAACGCAT  0 792 916030 1867 1882 25630 25645 AAGGGACCCTCTGCAC 38 793 916050 1891 1906 25654 25669 TAATAGGGCCACGAAA 53 794 916070 1921 1936 25684 25699 AAGAACCTCATGCTGG 24 795 916090 1964 1979 25727 25742 CCCCCATCACAAGGCC 24 796 916110 1986 2001 25749 25764 GATCACACATGGGCCA  0 797 916130 2081 2096 25844 25859 ACCTGACAGGCAGTGT 54 798 916149 2109 2124 25872 25887 GTAGTCAAGGTTATCA 87 799 916169 2154 2169 25917 25932 TAAGTAGATTATCATC 79 800 916189 2282 2297 26045 26060 AGGCTTATTCTACTAA 85 801 916209 2631 2646 26394 26409 GTGAATGCCCTGCACA 59 802 916229 2693 2708 26456 26471 GTGCTCCAGTGGAGAG 54 803 916249 2727 2742 26490 26505 GGTCCCTGCAGAAAAT 38 804 916289 N/A N/A  4338  4353 AAGCATACCACACCCC 79 805 916309 N/A N/A  5278  5293 AATCTTGGGATGCACA 95 806 916329 N/A N/A  5569  5584 CATCATGGCTTCCAGT 79 807 916349 N/A N/A  5879  5894 TGGGATGGTAATACTT  0 808 916369 N/A N/A  7134  7149 AAGTAAATGGAGTCCT  5 809 916389 N/A N/A  7615  7630 TTGCATAATCCCCTTA 33 810 916409 N/A N/A  8165  8180 TTAACTAGATCACTGA 58 811 916429 N/A N/A  9109  9124 TCCTAATGCGAGTCCC 86 812 916449 N/A N/A  9522  9537 TGCTGCTGGGTGCACT 45 813 916469 N/A N/A 10199 10214 GGTGATGACACAGCAT 94 814 916489 N/A N/A 10382 10397 GCCATGTACAACTTTT 52 815 916509 N/A N/A 11152 11167 TACAATTTGGACAGAG 71 816 916529 N/A N/A 11546 11561 ACCTATAGGAGTGCCC 35 817 916549 N/A N/A 12204 12219 TTATTTCCGTTAACCA 97 818 916569 N/A N/A 12672 12687 AGAATCATCTTAGTGG 94 819 916589 N/A N/A 12989 13004 CGGAATAAGCCTCCAC  0 820 916609 N/A N/A 13752 13767 GGCACTAAGTGTTAGA 57 821 916629 N/A N/A 14375 14390 TCTCACAAGGCTGGCA 84 822 916649 N/A N/A 15137 15152 GCCATACCGGCTCCCT 30 823 916669 N/A N/A 15691 15706 GGCCTTACAGAAACCA 15 824 916689 N/A N/A 16672 16687 AGTAATAGGCCAAGTC 16 825 916709 N/A N/A 17328 17343 ACAAACTAATGCACCT 42 826 916729 N/A N/A 18333 18348 TCCAATATCATAGCTG 32 827 916749 N/A N/A 19445 19460 CTACTAGGCATCTCTA 32 828 916768 N/A N/A 20553 20568 CTTACTATGGGATGAG 83 829 916788 N/A N/A 20808 20823 TAATATTCAGACCAGG 94 830 916808 N/A N/A 21252 21267 CCATGCATGGCACAGT  4 831 916828 N/A N/A 21968 21983 AGACAGGAATCCAACC  0 832 916848 N/A N/A 22767 22782 GGACATGACAGACTAA 96 833 916868 N/A N/A 23637 23652 GCAGACACAACAGCTT 40 834 916888 N/A N/A 24507 24522 CCATGCTACGGGCTCT  0 835

TABLE 13 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915351   33   48  2771  2786 GGCCAGGGCATTCCCA  0 836 915371   83   98  2821  2836 GCAGCTCCGCCCGGCG  2 837 915391  132  147  2870  2885 GGTTAGGATCTGGGTC 54 838 915411  222  237  2960  2975 GGTAGAAGCCCAGGAA 57 839 915431  321  336  3059  3074 CGACGCAGTGCAACGC 49 840 915451  368  383  5942  5957 TGAGAGGACCTGCAGA  0 841 915471  400  415  5974  5989 ATGTTCCGACTCCTGG 82 842 915491  437  452  6011  6026 GAACTTGCTTAAGTTG 23 843 915511  468  483  6042  6057 CCGGGAGGCATTTGCA  0 844 915531  498  513  6072  6087 TTTTGCCGGAGATGAG 84 845 915551  520  535  6094  6109 ACTCTGGTAAGAGAGA  5 846 915571  567  582  6141  6156 CGTCTTTGGACCGAAA 64 847 915611  708  723 11926 11941 CGGTGATGGTTGTTTT 98 848 915631  783  798 12001 12016 TGGTGATGTCCACATG  0 849 915651  835  850 12053 12068 AAAGCTCTCGAGAGAA 35 850 915671  890  905 13626 13641 ATCCAAATATCCTCGA 42 851 915691  971  986 16091 16106 AGGATCCATCCCTTCT  0 852 915711 1005 1020 16125 16140 GACTCATGTTTGCCCA 73 853 915731 1079 1094 16199 16214 GAGACGCAGGTGGTCT  0 854 915751 1140 1155 N/A N/A GTGCTGTAGCGAGCCT  0 855 915771 1202 1217 19042 19057 AATGGGTAGCAAGTTG 80 856 915791 1248 1263 19088 19103 ATTCCACAGGCAGGGT 37 857 915811 1327 1342 23732 23747 GTCACCCACTGCAACC 52 858 915831 1357 1372 23762 23777 CACATCAGCACTCGAG 29 859 915851 1429 1444 25192 25207 TCCTGCTCAGGTGTGC  2 860 915871 1513 1528 25276 25291 GACCTGAGGATGGACC 20 861 915891 1558 1573 25321 25336 TCAGCACCAGCAGGTA 68 862 915911 1620 1635 25383 25398 CGCCTCCTCAAGTGAC 48 863 915931 1652 1667 25415 25430 ACTTTAGCACCTCTGA 93 864 915951 1695 1710 25458 25473 GTCACTACACAGCAAT 84 865 915971 1776 1791 25539 25554 GCACAGGCGAAAGGTT 74 866 915991 1798 1813 25561 25576 TAGAGTTAAGTGCTGG 84 867 916011 1825 1840 25588 25603 CAGCTGAATTAACGCA  0 868 916031 1868 1883 25631 25646 TAAGGGACCCTCTGCA 54 869 916051 1892 1907 25655 25670 TTAATAGGGCCACGAA 75 870 916071 1922 1937 25685 25700 TAAGAACCTCATGCTG 56 871 916091 1965 1980 25728 25743 CCCCCCATCACAAGGC  9 872 916111 1987 2002 25750 25765 AGATCACACATGGGCC 26 873 916131 2084 2099 25847 25862 ACCACCTGACAGGCAG 80 874 916150 2110 2125 25873 25888 AGTAGTCAAGGTTATC 92 875 916170 2174 2189 25937 25952 TGAAAAAGGTGTTCTA 49 876 916190 2283 2298 26046 26061 AAGGCTTATTCTACTA 79 877 916210 2633 2648 26396 26411 AGGTGAATGCCCTGCA 71 878 916230 2694 2709 26457 26472 TGTGCTCCAGTGGAGA 75 879 916250 2728 2743 26491 26506 TGGTCCCTGCAGAAAA 79 880 916290 N/A N/A  4397  4412 TGCCTACTGGCTCACA 14 881 916310 N/A N/A  5279  5294 AAATCTTGGGATGCAC 94 882 916330 N/A N/A  5572  5587 TGACATCATGGCTTCC 93 883 916350 N/A N/A  6158  6173 GCTTACATCCACGACT  0 884 916370 N/A N/A  7135  7150 CAAGTAAATGGAGTCC 77 885 916390 N/A N/A  7620  7635 ATCTATTGCATAATCC 86 886 916550 N/A N/A 12205 12220 TTTATTTCCGTTAACC 96 887 916570 N/A N/A 12694 12709 TTCTTGACCGTGTTTC 98 888 916590 N/A N/A 12990 13005 CCGGAATAAGCCTCCA 47 889 916610 N/A N/A 13822 13837 TGTACAATGGGACGGA 69 890 916630 N/A N/A 14418 14433 ATCGACACAGCATCAC 92 891 916650 N/A N/A 15138 15153 TGCCATACCGGCTCCC  0 892 916670 N/A N/A 15758 15773 GGTTTATAACAACTGA 89 893 916690 N/A N/A 16722 16737 GCCTTGAGGTGGGTGG  0 894 916710 N/A N/A 17512 17527 AGTCATGGGATGTGCA 58 895 916730 N/A N/A 18395 18410 ATGTTTGGAAGTCGCC 92 896 916750 N/A N/A 19473 19488 AAGGATCCTGCTTCTA  9 897 916769 N/A N/A 20554 20569 GCTTACTATGGGATGA 62 898 916789 N/A N/A 20809 20824 GTAATATTCAGACCAG 96 899 916809 N/A N/A 21254 21269 ATCCATGCATGGCACA 72 900 916829 N/A N/A 21979 21994 GTCAGACACGGAGACA  0 901 916849 N/A N/A 23110 23125 GGCTTTTGAAGGAGAG 84 902 916869 N/A N/A 23787 23802 TATTTACCTGGAGGCG  0 903 916889 N/A N/A 24612 24627 CAAATCGGATCTTTGC 44 904

TABLE 14 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915352   34   49  2772  2787 CGGCCAGGGCATTCCC  0 905 915372   86  101  2824  2839 GCAGCAGCTCCGCCCG 48 906 915392  135  150  2873  2888 GCGGGTTAGGATCTGG 25 907 915412  225  240  2963  2978 CGTGGTAGAAGCCCAG  2 908 915432  322  337  3060  3075 CCGACGCAGTGCAACG 31 909 915452  371  386  5945  5960 ATCTGAGAGGACCTGC 72 910 915472  401  416  5975  5990 AATGTTCCGACTCCTG 77 911 915492  438  453  6012  6027 GGAACTTGCTTAAGTT 55 912 915512  469  484  6043  6058 GCCGGGAGGCATTTGC  3 913 915532  499  514  6073  6088 ATTTTGCCGGAGATGA 86 914 915552  521  536  6095  6110 CACTCTGGTAAGAGAG  8 915 915612  709  724 11927 11942 ACGGTGATGGTTGTTT 87 916 915632  787  802 12005 12020 AGCTTGGTGATGTCCA 38 917 915652  836  851 12054 12069 AAAAGCTCTCGAGAGA  0 918 915672  891  906 13627 13642 CATCCAAATATCCTCG 82 919 915692  972  987 16092 16107 CAGGATCCATCCCTTC  0 920 915712 1006 1021 16126 16141 AGACTCATGTTTGCCC 77 921 915732 1081 1096 16201 16216 CTGAGACGCAGGTGGT 87 922 915752 1141 1156 N/A N/A AGTGCTGTAGCGAGCC  1 923 915772 1203 1218 19043 19058 TAATGGGTAGCAAGTT 77 924 915792 1257 1272 19097 19112 CAATGGCAGATTCCAC 56 925 915812 1328 1343 23733 23748 GGTCACCCACTGCAAC 58 926 915832 1358 1373 23763 23778 ACACATCAGCACTCGA 66 927 915852 1430 1445 25193 25208 GTCCTGCTCAGGTGTG 52 928 915872 1518 1533 25281 25296 GGCTGGACCTGAGGAT  0 929 915892 1570 1585 25333 25348 GTGGAGAGCCCCTCAG 47 930 915912 1621 1636 25384 25399 TCGCCTCCTCAAGTGA  8 931 915932 1654 1669 25417 25432 AAACTTTAGCACCTCT 90 932 915952 1696 1711 25459 25474 GGTCACTACACAGCAA 82 933 915972 1777 1792 25540 25555 TGCACAGGCGAAAGGT 64 934 915992 1799 1814 25562 25577 TTAGAGTTAAGTGCTG 91 935 916012 1826 1841 25589 25604 CCAGCTGAATTAACGC 32 936 916032 1869 1884 25632 25647 GTAAGGGACCCTCTGC 73 937 916052 1894 1909 25657 25672 CATTAATAGGGCCACG 81 938 916072 1923 1938 25686 25701 CTAAGAACCTCATGCT 70 939 916092 1966 1981 25729 25744 ACCCCCCATCACAAGG 30 940 916112 1988 2003 25751 25766 AAGATCACACATGGGC 86 941 916132 2085 2100 25848 25863 GACCACCTGACAGGCA 61 942 916151 2111 2126 25874 25889 TAGTAGTCAAGGTTAT 88 943 916171 2176 2191 25939 25954 GGTGAAAAAGGTGTTC 84 944 916191 2284 2299 26047 26062 TAAGGCTTATTCTACT 76 945 916211 2634 2649 26397 26412 GAGGTGAATGCCCTGC  0 946 916231 2695 2710 26458 26473 GTGTGCTCCAGTGGAG 87 947 916251 2729 2744 26492 26507 CTGGTCCCTGCAGAAA 67 948 916291 N/A N/A  4419  4434 CAATGCTACTTGCCCC 68 949 916311 N/A N/A  5280  5295 TAAATCTTGGGATGCA 94 950 916331 N/A N/A  5576  5591 ACAATGACATCATGGC 97 951 916351 N/A N/A  6165  6180 GCAAACTGCTTACATC  0 952 916371 N/A N/A  7172  7187 GTTAGACGCGCCAGGC  7 953 916391 N/A N/A  7624  7639 TCTCATCTATTGCATA  0 954 916551 N/A N/A 12206 12221 TTTTATTTCCGTTAAC 73 955 916571 N/A N/A 12714 12729 TAAACTACCGAACGCA 96 956 916591 N/A N/A 12991 13006 CCCGGAATAAGCCTCC 47 957 916611 N/A N/A 13823 13838 CTGTACAATGGGACGG 23 958 916631 N/A N/A 14422 14437 TCCCATCGACACAGCA 95 959 916651 N/A N/A 15206 15221 GGAATATTGCCAGGTA 95 960 916671 N/A N/A 15759 15774 TGGTTTATAACAACTG 29 961 916691 N/A N/A 16746 16761 ATTAGGAGAGGTCTCA 55 962 916711 N/A N/A 17602 17617 CTTGATAGTGAATGTG 90 963 916731 N/A N/A 18859 18874 GGCACTCACAAAAGCG 10 964 916751 N/A N/A 20182 20197 CCCTATGTTCTACTTT 54 965 916770 N/A N/A 20572 20587 CAACATCTCTAGCTGG 82 966 916790 N/A N/A 20810 20825 GGTAATATTCAGACCA  0 967 916810 N/A N/A 21265 21280 TGAAGCTACAGATCCA 74 968 916830 N/A N/A 22042 22057 GGAAATCTGTCAGAGC 18 969 916850 N/A N/A 23142 23157 GAATCTAGGAAGGCGA 77 970 916870 N/A N/A 23789 23804 AGTATTTACCTGGAGG  0 971 916890 N/A N/A 24738 24753 AGCCTTAGGAAGCCTC 16 972

TABLE 15 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915353   35   50  2773  2788 TCGGCCAGGGCATTCC  0  973 915373   87  102  2825  2840 CGCAGCAGCTCCGCCC  0  974 915393  136  151  2874  2889 CGCGGGTTAGGATCTG  0  975 915413  239  254  2977  2992 GCGGGTCGCCCCGACG  0  976 915433  325  340  3063  3078 ACGCCGACGCAGTGCA  0  977 915453  372  387  5946  5961 GATCTGAGAGGACCTG 24  978 915473  402  417  5976  5991 CAATGTTCCGACTCCT 73  979 915493  441  456  6015  6030 GGAGGAACTTGCTTAA 87  980 915513  470  485  6044  6059 GGCCGGGAGGCATTTG  0  981 915533  500  515  6074  6089 TATTTTGCCGGAGATG 75  982 915553  522  537  6096  6111 ACACTCTGGTAAGAGA  0  983 915613  710  725 11928 11943 CACGGTGATGGTTGTT 64  984 915633  788  803 12006 12021 GAGCTTGGTGATGTCC 74  985 915653  837  852 12055 12070 CAAAAGCTCTCGAGAG  0  986 915673  892  907 13628 13643 GCATCCAAATATCCTC 81  987 915693  973  988 16093 16108 TCAGGATCCATCCCTT 10  988 915713 1007 1022 16127 16142 CAGACTCATGTTTGCC  0  989 915733 1082 1097 16202 16217 GCTGAGACGCAGGTGG 64  990 915753 1142 1157 N/A N/A CAGTGCTGTAGCGAGC  0  991 915773 1204 1219 19044 19059 CTAATGGGTAGCAAGT 72  992 915793 1258 1273 19098 19113 GCAATGGCAGATTCCA 57  993 915813 1329 1344 23734 23749 AGGTCACCCACTGCAA 56  994 915833 1359 1374 23764 23779 GACACATCAGCACTCG 43  995 915853 1431 1446 25194 25209 AGTCCTGCTCAGGTGT 66  996 915873 1525 1540 25288 25303 AAGTTCAGGCTGGACC 54  997 915893 1571 1586 25334 25349 GGTGGAGAGCCCCTCA  0  998 915913 1622 1637 25385 25400 CTCGCCTCCTCAAGTG 52  999 915933 1660 1675 25423 25438 GATGGGAAACTTTAGC 85 1000 915953 1697 1712 25460 25475 GGGTCACTACACAGCA 78 1001 915973 1778 1793 25541 25556 CTGCACAGGCGAAAGG 35 1002 915993 1800 1815 25563 25578 ATTAGAGTTAAGTGCT 63 1003 916013 1827 1842 25590 25605 ACCAGCTGAATTAACG 66 1004 916033 1873 1888 25636 25651 GTCAGTAAGGGACCCT 52 1005 916053 1897 1912 25660 25675 GACCATTAATAGGGCC 51 1006 916073 1924 1939 25687 25702 TCTAAGAACCTCATGC 55 1007 916093 1967 1982 25730 25745 TACCCCCCATCACAAG 15 1008 916113 1990 2005 25753 25768 ACAAGATCACACATGG 72 1009 916133 2086 2101 25849 25864 AGACCACCTGACAGGC 79 1010 916152 2112 2127 25875 25890 TTAGTAGTCAAGGTTA 84 1011 916172 2177 2192 25940 25955 AGGTGAAAAAGGTGTT 88 1012 916192 2285 2300 26048 26063 TTAAGGCTTATTCTAC 82 1013 916212 2635 2650 26398 26413 TGAGGTGAATGCCCTG 58 1014 916232 2696 2711 26459 26474 TGTGTGCTCCAGTGGA 89 1015 916252 2730 2745 26493 26508 GCTGGTCCCTGCAGAA 44 1016 916272 N/A N/A  3328  3343 GGGACGCACGAGAGTC  0 1017 916292 N/A N/A  4432  4447 GTCAATAGCTTCACAA 86 1018 916312 N/A N/A  5281  5296 ATAAATCTTGGGATGC 92 1019 916332 N/A N/A  5577  5592 CACAATGACATCATGG 95 1020 916352 N/A N/A  6170  6185 GATAAGCAAACTGCTT 19 1021 916372 N/A N/A  7192  7207 GAGGATGCAACTGGCT 84 1022 916392 N/A N/A  7644  7659 TCGGACTTCAGGCCCA  0 1023 916552 N/A N/A 12208 12223 CCTTTTATTTCCGTTA 97 1024 916572 N/A N/A 12745 12760 GCATACTAAAACCACC 85 1025 916592 N/A N/A 13375 13390 GACTTTGCAGGCACCC 92 1026 916612 N/A N/A 13909 13924 TGACATCCCAGTTCAA 30 1027 916632 N/A N/A 14427 14442 TACTTTCCCATCGACA 81 1028 916652 N/A N/A 15207 15222 AGGAATATTGCCAGGT 88 1029 916672 N/A N/A 15768 15783 GGTTAGTGTTGGTTTA 92 1030 916692 N/A N/A 16790 16805 CATTCGATGGAGGTTC 58 1031 916712 N/A N/A 17629 17644 GGCGGATTTCCCCACT 11 1032 916732 N/A N/A 18894 18909 TAAAATACGCCCGTCC  7 1033 916752 N/A N/A 20183 20198 TCCCTATGTTCTACTT 32 1034 916771 N/A N/A 20574 20589 ATCAACATCTCTAGCT 46 1035 916791 N/A N/A 20811 20826 GGGTAATATTCAGACC 43 1036 916811 N/A N/A 21313 21328 TTTACTAGAGACTCTG 69 1037 916831 N/A N/A 22071 22086 GTAGGATAGGACTAGA 45 1038 916851 N/A N/A 23219 23234 ATAAATGCCTGACCAC 64 1039 916871 N/A N/A 23861 23876 TGTTTCTAGAATGTCG 68 1040 916891 N/A N/A 24873 24888 GCCTATCAGTTTCCCC  0 1041

TABLE 16 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915354   36   51  2774  2789 CTCGGCCAGGGCATTC  0 1042 915374   89  104  2827  2842 TCCGCAGCAGCTCCGC 60 1043 915394  137  152  2875  2890 GCGCGGGTTAGGATCT  0 1044 915414  240  255  2978  2993 AGCGGGTCGCCCCGAC 21 1045 915434  337  352  3075  3090 ATACCGGAGAGGACGC 85 1046 915454  374  389  5948  5963 AAGATCTGAGAGGACC 24 1047 915474  403  418  5977  5992 CCAATGTTCCGACTCC 95 1048 915494  442  457  6016  6031 CGGAGGAACTTGCTTA 93 1049 915514  471  486  6045  6060 TGGCCGGGAGGCATTT  0 1050 915534  501  516  6075  6090 CTATTTTGCCGGAGAT 87 1051 915554  523  538  6097  6112 GACACTCTGGTAAGAG 26 1052 915614  711  726 11929 11944 ACACGGTGATGGTTGT 46 1053 915634  791  806 12009 12024 ACTGAGCTTGGTGATG 87 1054 915654  838  853 12056 12071 ACAAAAGCTCTCGAGA  0 1055 915674  900  915 13636 13651 ACCTGAATGCATCCAA 93 1056 915694  974  989 16094 16109 CTCAGGATCCATCCCT 43 1057 915714 1008 1023 16128 16143 CCAGACTCATGTTTGC  0 1058 915734 1083 1098 16203 16218 TGCTGAGACGCAGGTG 50 1059 915754 1143 1158 N/A N/A TCAGTGCTGTAGCGAG 42 1060 915774 1208 1223 19048 19063 TATCCTAATGGGTAGC 53 1061 915794 1260 1275 19100 19115 TCGCAATGGCAGATTC 67 1062 915814 1333 1348 23738 23753 TGTGAGGTCACCCACT  0 1063 915834 1360 1375 23765 23780 AGACACATCAGCACTC 24 1064 915854 1432 1447 25195 25210 CAGTCCTGCTCAGGTG 54 1065 915874 1526 1541 25289 25304 GAAGTTCAGGCTGGAC 75 1066 915894 1572 1587 25335 25350 AGGTGGAGAGCCCCTC  0 1067 915914 1623 1638 25386 25401 ACTCGCCTCCTCAAGT  0 1068 915934 1661 1676 25424 25439 AGATGGGAAACTTTAG 84 1069 915954 1724 1739 25487 25502 GGCTGGGATCCTCCAC 24 1070 915974 1779 1794 25542 25557 GCTGCACAGGCGAAAG 56 1071 915994 1801 1816 25564 25579 TATTAGAGTTAAGTGC 75 1072 916014 1828 1843 25591 25606 AACCAGCTGAATTAAC 55 1073 916034 1875 1890 25638 25653 CAGTCAGTAAGGGACC 70 1074 916054 1898 1913 25661 25676 TGACCATTAATAGGGC 74 1075 916074 1925 1940 25688 25703 TTCTAAGAACCTCATG 22 1076 916094 1968 1983 25731 25746 CTACCCCCCATCACAA  0 1077 916114 1992 2007 25755 25770 CCACAAGATCACACAT  0 1078 916134 2087 2102 25850 25865 CAGACCACCTGACAGG 78 1079 916153 2113 2128 25876 25891 TTTAGTAGTCAAGGTT 93 1080 916173 2178 2193 25941 25956 TAGGTGAAAAAGGTGT 89 1081 916193 2306 2321 26069 26084 ACCCAACCGATTTTTT 61 1082 916213 2636 2651 26399 26414 CTGAGGTGAATGCCCT 73 1083 916233 2697 2712 26460 26475 TTGTGTGCTCCAGTGG 92 1084 916253 2746 2761 26509 26524 TCACTGACCATGTGGG 16 1085 916273 N/A N/A  3362  3377 CTTCATGCACGGGCGC 37 1086 916293 N/A N/A  4462  4477 GCATAATCTCCTGCCT  0 1087 916313 N/A N/A  5284  5299 GCCATAAATCTTGGGA 37 1088 916333 N/A N/A  5605  5620 CTTTATTCAATGTGGC 97 1089 916353 N/A N/A  6529  6544 TACAACTGCCTGTGTT  0 1090 916373 N/A N/A  7218  7233 AAAGCTTCCGCAAACA 51 1091 916393 N/A N/A  7657  7672 CTAACATACACCCTCG  0 1092 916553 N/A N/A 12225 12240 AGCTTCTGGGACAAGC 10 1093 916573 N/A N/A 12746 12761 GGCATACTAAAACCAC 55 1094 916593 N/A N/A 13397 13412 TTGAATGTCACCCTTC 91 1095 916613 N/A N/A 13914 13929 AGTCATGACATCCCAG 93 1096 916633 N/A N/A 14442 14457 TCTCATTGGCACCTGT 86 1097 916653 N/A N/A 15252 15267 CCCTATCAGATGCCCT 81 1098 916673 N/A N/A 15799 15814 CATATCTGGTTTCATG  0 1099 916693 N/A N/A 16842 16857 GACCATAGCACTGTCT  0 1100 916713 N/A N/A 17737 17752 ATTAATCTGGTCATAT  0 1101 916733 N/A N/A 18898 18913 TCCATAAAATACGCCC 69 1102 916753 N/A N/A 20195 20210 GAAAGATGGAATTCCC 86 1103 916772 N/A N/A 20604 20619 TACGATCATCATTATT 91 1104 916792 N/A N/A 20841 20856 GTATTAGCTCAATATT  0 1105 916812 N/A N/A 21314 21329 GTTTACTAGAGACTCT 64 1106 916832 N/A N/A 22080 22095 GTAAAAACTGTAGGAT  0 1107 916852 N/A N/A 23220 23235 GATAAATGCCTGACCA 29 1108 916872 N/A N/A 24011 24026 CCGACGGGAAGTCTTC  0 1109 916892 N/A N/A 24874 24889 GGCCTATCAGTTTCCC  0 1110

TABLE 17 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915355   37   52  2775  2790 TCTCGGCCAGGGCATT  0 1111 915375   90  105  2828  2843 ATCCGCAGCAGCTCCG 52 1112 915395  138  153  2876  2891 GGCGCGGGTTAGGATC  0 1113 915415  241  256  2979  2994 CAGCGGGTCGCCCCGA  8 1114 915435  338  353  3076  3091 GATACCGGAGAGGACG 30 1115 915455  378  393  5952  5967 GCACAAGATCTGAGAG 72 1116 915475  405  420  5979  5994 TGCCAATGTTCCGACT  0 1117 915495  443  458  6017  6032 TCGGAGGAACTTGCTT 69 1118 915515  472  487  6046  6061 TTGGCCGGGAGGCATT  9 1119 915535  502  517  6076  6091 CCTATTTTGCCGGAGA 96 1120 915555  524  539  6098  6113 AGACACTCTGGTAAGA  2 1121 915615  712  727 11930 11945 GACACGGTGATGGTTG 32 1122 915635  792  807 12010 12025 GACTGAGCTTGGTGAT 93 1123 915655  839  854 12057 12072 GACAAAAGCTCTCGAG 40 1124 915675  901  916 13637 13652 AACCTGAATGCATCCA 92 1125 915695  975  990 16095 16110 CCTCAGGATCCATCCC  0 1126 915715 1011 1026 16131 16146 AATCCAGACTCATGTT 67 1127 915735 1088 1103 16208 16223 CAGGATGCTGAGACGC 86 1128 915755 1144 1159 N/A N/A CTCAGTGCTGTAGCGA 25 1129 915775 1209 1224 19049 19064 TTATCCTAATGGGTAG 23 1130 915795 1261 1276 19101 19116 ATCGCAATGGCAGATT  0 1131 915815 1337 1352 23742 23757 CACCTGTGAGGTCACC 35 1132 915835 1361 1376 23766 23781 CAGACACATCAGCACT 54 1133 915855 1433 1448 25196 25211 CCAGTCCTGCTCAGGT 23 1134 915875 1530 1545 25293 25308 AGAAGAAGTTCAGGCT 81 1135 915895 1574 1589 25337 25352 AAAGGTGGAGAGCCCC 76 1136 915915 1624 1639 25387 25402 GACTCGCCTCCTCAAG 75 1137 915935 1674 1689 25437 25452 GGTAGCTGCACAAAGA 76 1138 915955 1726 1741 25489 25504 GAGGCTGGGATCCTCC  0 1139 915975 1780 1795 25543 25558 CGCTGCACAGGCGAAA  0 1140 915995 1805 1820 25568 25583 GATGTATTAGAGTTAA 82 1141 916015 1829 1844 25592 25607 CAACCAGCTGAATTAA 59 1142 916035 1876 1891 25639 25654 ACAGTCAGTAAGGGAC 81 1143 916055 1899 1914 25662 25677 CTGACCATTAATAGGG 49 1144 916075 1929 1944 25692 25707 GTCATTCTAAGAACCT 81 1145 916095 1969 1984 25732 25747 CCTACCCCCCATCACA 21 1146 916115 1995 2010 25758 25773 ACCCCACAAGATCACA  0 1147 916135 2088 2103 25851 25866 GCAGACCACCTGACAG 44 1148 916154 2131 2146 25894 25909 CCCCGCCATGGAGACG 68 1149 916174 2180 2195 25943 25958 GTTAGGTGAAAAAGGT 90 1150 916194 2308 2323 26071 26086 GCACCCAACCGATTTT 83 1151 916214 2637 2652 26400 26415 GCTGAGGTGAATGCCC 52 1152 916234 2698 2713 26461 26476 GTTGTGTGCTCCAGTG 88 1153 916254 2747 2762 26510 26525 CTCACTGACCATGTGG 13 1154 916274 N/A N/A  3524  3539 GCAAATCGGCCCCTCG  3 1155 916294 N/A N/A  4463  4478 GGCATAATCTCCTGCC  0 1156 916314 N/A N/A  5324  5339 TGGCATGCAAGACCAC  0 1157 916334 N/A N/A  5606  5621 ACTTTATTCAATGTGG 95 1158 916354 N/A N/A  6556  6571 GTTTATGTCACTCTGG 68 1159 916374 N/A N/A  7245  7260 GAACAGACAAGTGCTG 38 1160 916394 N/A N/A  7658  7673 ACTAACATACACCCTC 31 1161 916554 N/A N/A 12249 12264 ATAATCAGGGTGGTGC  0 1162 916574 N/A N/A 12747 12762 AGGCATACTAAAACCA 47 1163 916594 N/A N/A 13500 13515 GAATCATGCAAGCTCT 50 1164 916614 N/A N/A 13996 14011 TAAACTAAGGGTCACA 37 1165 916634 N/A N/A 14497 14512 ATCCATCCTGCATGAG 76 1166 916654 N/A N/A 15254 15269 GGCCCTATCAGATGCC  0 1167 916674 N/A N/A 15802 15817 CTACATATCTGGTTTC  0 1168 916694 N/A N/A 16844 16859 TGGACCATAGCACTGT 60 1169 916714 N/A N/A 17738 17753 TATTAATCTGGTCATA 18 1170 916734 N/A N/A 18926 18941 CCACTTTACTCTGTTG 64 1171 916754 N/A N/A 20210 20225 AACTATGCCTAGAACG 43 1172 916773 N/A N/A 20606 20621 TTTACGATCATCATTA 77 1173 916793 N/A N/A 20842 20857 TGTATTAGCTCAATAT  0 1174 916813 N/A N/A 21319 21334 TGGGAGTTTACTAGAG 66 1175 916833 N/A N/A 22118 22133 AGAGAGTACTCTTGGA 11 1176 916853 N/A N/A 23222 23237 CTGATAAATGCCTGAC 78 1177 916873 N/A N/A 24038 24053 ATCAATGCTGCACTCA 88 1178 916893 N/A N/A 24889 24904 ACGAATCCCTGGAGGG  0 1179

TABLE 18 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915356   38   53  2776  2791 GTCTCGGCCAGGGCAT  0 1180 915376   93  108  2831  2846 CTGATCCGCAGCAGCT 28 1181 915396  165  180  2903  2918 CGTACATGGCGGCGGC  0 1182 915416  242  257  2980  2995 GCAGCGGGTCGCCCCG  0 1183 915436  339  354  3077  3092 GGATACCGGAGAGGAC 64 1184 915456  379  394  5953  5968 CGCACAAGATCTGAGA 79 1185 915476  406  421  5980  5995 ATGCCAATGTTCCGAC 83 1186 915496  444  459  6018  6033 GTCGGAGGAACTTGCT 35 1187 915516  473  488  6047  6062 ATTGGCCGGGAGGCAT  0 1188 915536  503  518  6077  6092 GCCTATTTTGCCGGAG 77 1189 915556  525  540  6099  6114 CAGACACTCTGGTAAG 62 1190 915616  730  745 11948 11963 TACTCCCCATAGAAGG  8 1191 915636  794  809 12012 12027 TAGACTGAGCTTGGTG 90 1192 915656  840  855 12058 12073 GGACAAAAGCTCTCGA 60 1193 915676  902  917 13638 13653 GAACCTGAATGCATCC 72 1194 915696  978  993 16098 16113 CGACCTCAGGATCCAT  0 1195 915716 1012 1027 16132 16147 GAATCCAGACTCATGT  0 1196 915736 1089 1104 16209 16224 GCAGGATGCTGAGACG 55 1197 915756 1145 1160 N/A N/A ACTCAGTGCTGTAGCG 12 1198 915776 1210 1225 19050 19065 ATTATCCTAATGGGTA 28 1199 915796 1262 1277 19102 19117 AATCGCAATGGCAGAT  0 1200 915816 1339 1354 23744 23759 AACACCTGTGAGGTCA 56 1201 915836 1365 1380 23770 23785 GGAGCAGACACATCAG 53 1202 915856 1434 1449 25197 25212 GCCAGTCCTGCTCAGG 21 1203 915876 1531 1546 25294 25309 AAGAAGAAGTTCAGGC 85 1204 915896 1575 1590 25338 25353 GAAAGGTGGAGAGCCC 78 1205 915916 1626 1641 25389 25404 TAGACTCGCCTCCTCA 32 1206 915936 1676 1691 25439 25454 GAGGTAGCTGCACAAA 91 1207 915956 1737 1752 25500 25515 AACTCAGCTCAGAGGC 46 1208 915976 1781 1796 25544 25559 CCGCTGCACAGGCGAA  0 1209 915996 1807 1822 25570 25585 CTGATGTATTAGAGTT 93 1210 916016 1830 1845 25593 25608 CCAACCAGCTGAATTA 21 1211 916036 1877 1892 25640 25655 AACAGTCAGTAAGGGA 82 1212 916056 1900 1915 25663 25678 TCTGACCATTAATAGG 13 1213 916076 1930 1945 25693 25708 TGTCATTCTAAGAACC 40 1214 916096 1970 1985 25733 25748 GCCTACCCCCCATCAC 18 1215 916116 1996 2011 25759 25774 CACCCCACAAGATCAC 50 1216 916136 2089 2104 25852 25867 TGCAGACCACCTGACA 58 1217 916155 2132 2147 25895 25910 CCCCCGCCATGGAGAC 33 1218 916175 2224 2239 25987 26002 CGCTTCCTTACATTTT 89 1219 916195 2309 2324 26072 26087 TGCACCCAACCGATTT 64 1220 916215 2638 2653 26401 26416 GGCTGAGGTGAATGCC  0 1221 916235 2699 2714 26462 26477 AGTTGTGTGCTCCAGT 85 1222 916255 2748 2763 26511 26526 ACTCACTGACCATGTG  0 1223 916275 N/A N/A  3555  3570 GGCCAAAGCCCCACTC  0 1224 916295 N/A N/A  4464  4479 GGGCATAATCTCCTGC  0 1225 916315 N/A N/A  5342  5357 GGCTGATCTGCACTCT 84 1226 916335 N/A N/A  5626  5641 TAATTCTACCTGTGTC 92 1227 916355 N/A N/A  6557  6572 AGTTTATGTCACTCTG 27 1228 916375 N/A N/A  7321  7336 ACACTTTGCGAAGCAC 27 1229 916395 N/A N/A  7660  7675 GAACTAACATACACCC  1 1230 916555 N/A N/A 12252 12267 CCCATAATCAGGGTGG  0 1231 916575 N/A N/A 12758 12773 GTAGAGTGGTAAGGCA 95 1232 916595 N/A N/A 13502 13517 AAGAATCATGCAAGCT 34 1233 916615 N/A N/A 13997 14012 TTAAACTAAGGGTCAC 65 1234 916635 N/A N/A 14549 14564 TTAATGTGGATTCACG 76 1235 916655 N/A N/A 15295 15310 CCAAGATAACCTCACA 64 1236 916675 N/A N/A 15806 15821 CCATCTACATATCTGG 26 1237 916695 N/A N/A 16854 16869 CACAATCATTTGGACC 72 1238 916715 N/A N/A 17739 17754 GTATTAATCTGGTCAT 87 1239 916735 N/A N/A 19113 19128 CACCTCTGGACAATCG 29 1240 916755 N/A N/A 20212 20227 CAAACTATGCCTAGAA 70 1241 916774 N/A N/A 20608 20623 ATTTTACGATCATCAT 91 1242 916794 N/A N/A 20846 20861 TGCCTGTATTAGCTCA 90 1243 916814 N/A N/A 21345 21360 CACATAAAGTCAAACG 87 1244 916834 N/A N/A 22124 22139 AGAACAAGAGAGTACT  3 1245 916854 N/A N/A 23250 23265 CACATAAAGGACCCCC 54 1246 916874 N/A N/A 24126 24141 CGCTATCTGACACTCC 87 1247 916894 N/A N/A 24896 24911 TCCACCAACGAATCCC 50 1248

TABLE 19 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915357   39   54  2777  2792 TGTCTCGGCCAGGGCA  0 1249 915377   94  109  2832  2847 CCTGATCCGCAGCAGC  0 1250 915397  182  197  2920  2935 CCAGCCGCGCTCTGCG  0 1251 915417  243  258  2981  2996 GGCAGCGGGTCGCCCC  0 1252 915437  341  356  3079  3094 CGGGATACCGGAGAGG 57 1253 915457  380  395  5954  5969 CCGCACAAGATCTGAG 71 1254 915477  407  422  5981  5996 GATGCCAATGTTCCGA 93 1255 915497  445  460  6019  6034 TGTCGGAGGAACTTGC 85 1256 915517  474  489  6048  6063 CATTGGCCGGGAGGCA  0 1257 915537  504  519  6078  6093 TGCCTATTTTGCCGGA 44 1258 915557  526  541  6100  6115 TCAGACACTCTGGTAA 26 1259 915617  732  747 11950 11965 CGTACTCCCCATAGAA 66 1260 915637  796  811 12014 12029 CGTAGACTGAGCTTGG 98 1261 915657  857  872 12075 12090 CACCTTGAGATCCGGG  0 1262 915677  903  918 13639 13654 AGAACCTGAATGCATC 78 1263 915697  979  994 16099 16114 GCGACCTCAGGATCCA  1 1264 915717 1031 1046 16151 16166 GGCAGCCGACTCCGGG 19 1265 915737 1109 1124 16229 16244 CAGGATGCTCTCATCC  0 1266 915757 1146 1161 N/A N/A CACTCAGTGCTGTAGC 33 1267 915777 1214 1229 19054 19069 AGACATTATCCTAATG 42 1268 915797 1263 1278 19103 19118 CAATCGCAATGGCAGA 49 1269 915817 1340 1355 23745 23760 GAACACCTGTGAGGTC 44 1270 915837 1398 1413 25161 25176 GGCTGCTCACTGGCAT 18 1271 915857 1435 1450 25198 25213 GGCCAGTCCTGCTCAG  0 1272 915877 1534 1549 25297 25312 CCCAAGAAGAAGTTCA 76 1273 915897 1576 1591 25339 25354 GGAAAGGTGGAGAGCC 24 1274 915917 1627 1642 25390 25405 CTAGACTCGCCTCCTC 77 1275 915937 1679 1694 25442 25457 GCGGAGGTAGCTGCAC 16 1276 915957 1738 1753 25501 25516 CAACTCAGCTCAGAGG 61 1277 915977 1782 1797 25545 25560 ACCGCTGCACAGGCGA 34 1278 915997 1809 1824 25572 25587 TGCTGATGTATTAGAG 83 1279 916017 1831 1846 25594 25609 CCCAACCAGCTGAATT 42 1280 916037 1878 1893 25641 25656 AAACAGTCAGTAAGGG 92 1281 916057 1901 1916 25664 25679 GTCTGACCATTAATAG 64 1282 916077 1931 1946 25694 25709 CTGTCATTCTAAGAAC 41 1283 916097 1971 1986 25734 25749 AGCCTACCCCCCATCA  0 1284 916117 1997 2012 25760 25775 CCACCCCACAAGATCA  0 1285 916137 2090 2105 25853 25868 TTGCAGACCACCTGAC 65 1286 916156 2133 2148 25896 25911 ACCCCCGCCATGGAGA 54 1287 916176 2225 2240 25988 26003 ACGCTTCCTTACATTT 84 1288 916196 2310 2325 26073 26088 CTGCACCCAACCGATT 58 1289 916216 2639 2654 26402 26417 GGGCTGAGGTGAATGC 46 1290 916236 2700 2715 26463 26478 AAGTTGTGTGCTCCAG 86 1291 916256 2751 2766 26514 26529 GAAACTCACTGACCAT 41 1292 916276 N/A N/A  4068  4083 GGAAACAACTTTCCTC  0 1293 916296 N/A N/A  4730  4745 GATCATGTGGCGGTCT 68 1294 916316 N/A N/A  5364  5379 CACTTACTGGCCTGGC 30 1295 916336 N/A N/A  5645  5660 ATATTGGGCTCAATGA 89 1296 916356 N/A N/A  6575  6590 ATCACTGGAGGTGTAC  0 1297 916376 N/A N/A  7328  7343 CAGGATCACACTTTGC 17 1298 916396 N/A N/A  7661  7676 GGAACTAACATACACC  0 1299 916556 N/A N/A 12272 12287 GTATATGTTCCCAGGT 81 1300 916576 N/A N/A 12788 12803 GTGTACATGGTCTGCA 94 1301 916596 N/A N/A 13529 13544 ATCATTGGAAGACCGC 89 1302 916616 N/A N/A 13998 14013 GTTAAACTAAGGGTCA 85 1303 916636 N/A N/A 14550 14565 CTTAATGTGGATTCAC 91 1304 916656 N/A N/A 15351 15366 TCCAACTTCAGGCTGA 74 1305 916676 N/A N/A 15819 15834 AGCTTTGTGGGCTCCA 69 1306 916696 N/A N/A 16982 16997 GTTTAATAAGGGCACC 63 1307 916716 N/A N/A 17740 17755 CGTATTAATCTGGTCA 93 1308 916736 N/A N/A 19126 19141 CACCTAAAATGCTCAC 17 1309 916756 N/A N/A 20213 20228 ACAAACTATGCCTAGA 58 1310 916775 N/A N/A 20609 20624 AATTTTACGATCATCA 78 1311 916795 N/A N/A 20927 20942 GACAGATCAGCACTCG 80 1312 916815 N/A N/A 21407 21422 CAATTCTAGACATGGC 88 1313 916835 N/A N/A 22338 22353 TGCACCTACCCTTTTC 39 1314 916855 N/A N/A 23251 23266 ACACATAAAGGACCCC 48 1315 916875 N/A N/A 24241 24256 GCATTACCAGGCACCT 61 1316 916895 N/A N/A 24912 24927 GACATCACAGGTGTTG  5 1317

TABLE 20 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915358   40   55  2778  2793 GTGTCTCGGCCAGGGC  0 1318 915378   96  111  2834  2849 GTCCTGATCCGCAGCA  0 1319 915398  184  199  2922  2937 CTCCAGCCGCGCTCTG 14 1320 915418  244  259  2982  2997 AGGCAGCGGGTCGCCC  0 1321 915438  342  357  3080  3095 GCGGGATACCGGAGAG 44 1322 915458  381  396  5955  5970 TCCGCACAAGATCTGA 41 1323 915478  408  423  5982  5997 AGATGCCAATGTTCCG 95 1324 915498  446  461  6020  6035 CTGTCGGAGGAACTTG 40 1325 915518  475  490  6049  6064 ACATTGGCCGGGAGGC  5 1326 915538  505  520  6079  6094 ATGCCTATTTTGCCGG 61 1327 915558  527  542  6101  6116 ATCAGACACTCTGGTA  0 1328 915618  748  763 11966 11981 ACTTTAGGGCAGATGT 87 1329 915638  818  833 12036 12051 GTAGAGGTTCCCTGTG 87 1330 915658  859  874 N/A N/A AGCACCTTGAGATCCG  0 1331 915678  926  941 N/A N/A GTTGCAGATGCCCTTC 24 1332 915698  980  995 16100 16115 GGCGACCTCAGGATCC  0 1333 915718 1032 1047 16152 16167 AGGCAGCCGACTCCGG  8 1334 915738 1114 1129 16234 16249 GTGTCCAGGATGCTCT 41 1335 915758 1147 1162 N/A N/A TCACTCAGTGCTGTAG 59 1336 915778 1217 1232 19057 19072 ATAAGACATTATCCTA 85 1337 915798 1264 1279 19104 19119 ACAATCGCAATGGCAG 66 1338 915818 1342 1357 23747 23762 GTGAACACCTGTGAGG 58 1339 915838 1400 1415 25163 25178 TTGGCTGCTCACTGGC 79 1340 915858 1436 1451 25199 25214 GGGCCAGTCCTGCTCA  0 1341 915878 1535 1550 25298 25313 GCCCAAGAAGAAGTTC 54 1342 915898 1590 1605 25353 25368 CTAGTGAAAAACTGGG 34 1343 915918 1628 1643 25391 25406 GCTAGACTCGCCTCCT 33 1344 915938 1680 1695 25443 25458 TGCGGAGGTAGCTGCA  0 1345 915958 1756 1771 25519 25534 CCTAGCTTTTCATAAA  0 1346 915978 1783 1798 25546 25561 GACCGCTGCACAGGCG 24 1347 915998 1810 1825 25573 25588 ATGCTGATGTATTAGA 86 1348 916018 1832 1847 25595 25610 TCCCAACCAGCTGAAT  3 1349 916038 1879 1894 25642 25657 GAAACAGTCAGTAAGG 64 1350 916058 1902 1917 25665 25680 AGTCTGACCATTAATA 86 1351 916078 1933 1948 25696 25711 ACCTGTCATTCTAAGA 18 1352 916098 1972 1987 25735 25750 CAGCCTACCCCCCATC 41 1353 916118 1999 2014 25762 25777 CTCCACCCCACAAGAT  0 1354 916138 2092 2107 25855 25870 CTTTGCAGACCACCTG 65 1355 916157 2134 2149 25897 25912 TACCCCCGCCATGGAG 57 1356 916177 2237 2252 26000 26015 CAACAGGTAACAACGC 88 1357 916197 2579 2594 26342 26357 GTCAGACTTTCACTCA 81 1358 916217 2659 2674 26422 26437 GTGCTTGGCTCCTGCC 43 1359 916237 2701 2716 26464 26479 CAAGTTGTGTGCTCCA 73 1360 916257 2769 2784 26532 26547 CATCGCCACACATGGG 61 1361 916277 N/A N/A  4105  4120 AGGAAGGGTCCCAAAC  0 1362 916297 N/A N/A  4731  4746 TGATCATGTGGCGGTC 80 1363 916317 N/A N/A  5391  5406 TGCTATCAGGTGCAGG 60 1364 916337 N/A N/A  5646  5661 TATATTGGGCTCAATG 71 1365 916357 N/A N/A  6594  6609 GTTTACAAACATGGAC 26 1366 916377 N/A N/A  7464  7479 TCATTAGCATCACCGG 33 1367 916397 N/A N/A  7662  7677 GGGAACTAACATACAC  0 1368 916557 N/A N/A 12274 12289 GGGTATATGTTCCCAG  0 1369 916577 N/A N/A 12830 12845 TGCATAGCCTTCTTTC 84 1370 916597 N/A N/A 13530 13545 CATCATTGGAAGACCG 62 1371 916617 N/A N/A 14016 14031 TCTTTAACTTCGGCCC 70 1372 916637 N/A N/A 14551 14566 TCTTAATGTGGATTCA 88 1373 916657 N/A N/A 15388 15403 TCAGACAACCACAGCT 66 1374 916677 N/A N/A 15852 15867 TAAAGCAGGACACACG 74 1375 916697 N/A N/A 17077 17092 AGACATGTTGGTGTCT  0 1376 916717 N/A N/A 17788 17803 CCCCAGTCTTTTATTC  0 1377 916737 N/A N/A 19140 19155 GGAAGACACGGAGCCA 20 1378 916757 N/A N/A 20240 20255 CCTAACTGCTGGCTCT 85 1379 916776 N/A N/A 20610 20625 TAATTTTACGATCATC 76 1380 916796 N/A N/A 20939 20954 CTCTTTGTAGCAGACA 90 1381 916816 N/A N/A 21439 21454 CAATATACTGAGAGGA 92 1382 916836 N/A N/A 22392 22407 GTAGACATCCTTCCCG 65 1383 916856 N/A N/A 23252 23267 GACACATAAAGGACCC 59 1384 916876 N/A N/A 24242 24257 TGCATTACCAGGCACC 37 1385 916896 N/A N/A 24913 24928 GGACATCACAGGTGTT 19 1386

TABLE 21 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915359   41   56  2779  2794 AGTGTCTCGGCCAGGG  0 1387 915379   97  112  2835  2850 GGTCCTGATCCGCAGC  0 1388 915399  185  200  2923  2938 GCTCCAGCCGCGCTCT 26 1389 915419  245  260  2983  2998 CAGGCAGCGGGTCGCC  0 1390 915439  343  358  3081  3096 AGCGGGATACCGGAGA 69 1391 915459  382  397  5956  5971 TTCCGCACAAGATCTG 71 1392 915479  409  424  5983  5998 AAGATGCCAATGTTCC 93 1393 915499  448  463  6022  6037 CCCTGTCGGAGGAACT 30 1394 915519  477  492  6051  6066 GGACATTGGCCGGGAG 88 1395 915539  506  521  6080  6095 GATGCCTATTTTGCCG 60 1396 915559  529  544  6103  6118 CCATCAGACACTCTGG 30 1397 915579  595  610  7825  7840 CAGGAACATACCAAGG 98 1398 915599  674  689 11892 11907 GTTGTCACTCACTCCT 98 1399 915619  749  764 11967 11982 GACTTTAGGGCAGATG 96 1400 915639  819  834 12037 12052 GGTAGAGGTTCCCTGT 92 1401 915659  860  875 N/A N/A CAGCACCTTGAGATCC  0 1402 915679  928  943 N/A N/A CTGTTGCAGATGCCCT 58 1403 915699  981  996 16101 16116 TGGCGACCTCAGGATC 40 1404 915719 1033 1048 16153 16168 AAGGCAGCCGACTCCG  0 1405 915739 1115 1130 16235 16250 GGTGTCCAGGATGCTC 40 1406 915759 1148 1163 N/A N/A TTCACTCAGTGCTGTA 29 1407 915779 1219 1234 19059 19074 ACATAAGACATTATCC 86 1408 915799 1268 1283 19108 19123 CTGGACAATCGCAATG 42 1409 915819 1344 1359 23749 23764 GAGTGAACACCTGTGA 81 1410 915839 1401 1416 25164 25179 GTTGGCTGCTCACTGG 85 1411 915859 1437 1452 25200 25215 AGGGCCAGTCCTGCTC  0 1412 915879 1538 1553 25301 25316 ATTGCCCAAGAAGAAG 54 1413 915899 1591 1606 25354 25369 TCTAGTGAAAAACTGG  0 1414 915919 1629 1644 25392 25407 TGCTAGACTCGCCTCC 72 1415 915939 1681 1696 25444 25459 ATGCGGAGGTAGCTGC 39 1416 915959 1764 1779 25527 25542 GGTTGCTTCCTAGCTT 87 1417 915979 1784 1799 25547 25562 GGACCGCTGCACAGGC  0 1418 915999 1811 1826 25574 25589 CATGCTGATGTATTAG 35 1419 916019 1833 1848 25596 25611 TTCCCAACCAGCTGAA  0 1420 916039 1880 1895 25643 25658 CGAAACAGTCAGTAAG 80 1421 916059 1905 1920 25668 25683 AACAGTCTGACCATTA 85 1422 916079 1934 1949 25697 25712 CACCTGTCATTCTAAG 45 1423 916099 1973 1988 25736 25751 CCAGCCTACCCCCCAT 53 1424 916119 2022 2037 25785 25800 GTGGGATCATGCTATT 76 1425 916139 2093 2108 25856 25871 TCTTTGCAGACCACCT 85 1426 916158 2135 2150 25898 25913 TTACCCCCGCCATGGA  0 1427 916178 2238 2253 26001 26016 TCAACAGGTAACAACG 87 1428 916198 2620 2635 26383 26398 GCACACTAGATTATTT 66 1429 916218 2673 2688 26436 26451 CGGAAGCTCCTGCTGT 27 1430 916238 2702 2717 26465 26480 TCAAGTTGTGTGCTCC 91 1431 916258 2770 2785 26533 26548 TCATCGCCACACATGG 49 1432 916278 N/A N/A  4211  4226 TCATTTCCAGGAGTAC 75 1433 916298 N/A N/A  4735  4750 CAAATGATCATGTGGC 93 1434 916318 N/A N/A  5394  5409 TAATGCTATCAGGTGC 95 1435 916338 N/A N/A  5648  5663 GATATATTGGGCTCAA 97 1436 916358 N/A N/A  6596  6611 GGGTTTACAAACATGG 75 1437 916378 N/A N/A  7465  7480 TTCATTAGCATCACCG 78 1438 916398 N/A N/A  7686  7701 GTTAATCCATGGGTCA 49 1439 916418 N/A N/A  8992  9007 AGCCTAAACTTCCTCC 63 1440 916438 N/A N/A  9318  9333 AGAAGAGCCGCCCTGC 77 1441 916458 N/A N/A  9795  9810 GCAAGACTAGCAAGTG 85 1442 916478 N/A N/A 10301 10316 AGCATGCGGTATGTAC 67 1443 916498 N/A N/A 10849 10864 CACACAATTTCTAGGG 82 1444 916518 N/A N/A 11346 11361 TTGACAATTAGAACCA 96 1445 916538 N/A N/A 11711 11726 ACAAATCCTTACCGAG 54 1446 916558 N/A N/A 12285 12300 GTTTTAGGTCTGGGTA 94 1447 916578 N/A N/A 12831 12846 TTGCATAGCCTTCTTT 93 1448 916598 N/A N/A 13660 13675 CATACATACCCTTCTC  9 1449 916618 N/A N/A 14025 14040 CGCAGAAACTCTTTAA 89 1450 916638 N/A N/A 14552 14567 GTCTTAATGTGGATTC 93 1451 916658 N/A N/A 15421 15436 AGCATTGGCACACTGG 70 1452 916678 N/A N/A 15857 15872 GGCTTTAAAGCAGGAC 62 1453 916698 N/A N/A 17079 17094 GCAGACATGTTGGTGT  2 1454 916718 N/A N/A 17839 17854 TACAAGCTGGTCCTTG  0 1455 916738 N/A N/A 19211 19226 GACAATCCAGGTCCCA 70 1456 916758 N/A N/A 20285 20300 GAGGAAGCCCAATCAA 81 1457 916777 N/A N/A 20611 20626 CTAATTTTACGATCAT 81 1458 916797 N/A N/A 20984 20999 TTAAACTGCCAAGTCC 83 1459 916817 N/A N/A 21440 21455 CCAATATACTGAGAGG 96 1460 916837 N/A N/A 22406 22421 GGTAGCACCGCCAAGT  0 1461 916857 N/A N/A 23301 23316 CACCATGGAGAGGTCT  0 1462 916877 N/A N/A 24243 24258 TTGCATTACCAGGCAC 17 1463 916897 N/A N/A 24934 24949 GCTACCTGGACACCTC 47 1464

TABLE 22 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 841947 2094 2109 25857 25872 ATCTTTGCAGACCACC 89 1464 912986 N/A N/A 20288 20303 TCAGAGGAAGCCCAAT 92  254 20318 20333 915360   42   57  2780  2795 CAGTGTCTCGGCCAGG  0 1466 915380   98  113  2836  2851 GGGTCCTGATCCGCAG  0 1467 915400  186  201  2924  2939 AGCTCCAGCCGCGCTC  0 1468 915420  246  261  2984  2999 TCAGGCAGCGGGTCGC 78 1469 915440  344  359  3082  3097 CAGCGGGATACCGGAG 72 1470 915460  383  398  5957  5972 CTTCCGCACAAGATCT  0 1471 915480  411  426  5985  6000 GGAAGATGCCAATGTT 94 1472 915500  449  464  6023  6038 ACCCTGTCGGAGGAAC 40 1473 915520  480  495  6054  6069 GGTGGACATTGGCCGG 38 1474 915540  507  522  6081  6096 AGATGCCTATTTTGCC 76 1475 915560  556  571  6130  6145 CGAAAGTCAGACACCA 69 1476 915620  750  765 11968 11983 TGACTTTAGGGCAGAT 89 1477 915640  821  836 12039 12054 AAGGTAGAGGTTCCCT 10 1478 915660  875  890 13611 13626 AAGGCATATCTCTCCC 47 1479 915680  929  944 N/A N/A CCTGTTGCAGATGCCC 22 1480 915700  982  997 16102 16117 ATGGCGACCTCAGGAT 58 1481 915720 1034 1049 16154 16169 CAAGGCAGCCGACTCC 63 1482 915740 1121 1136 16241 16256 CGAGAGGGTGTCCAGG  0 1483 915760 1149 1164 N/A N/A CTTCACTCAGTGCTGT 13 1484 915780 1226 1241 19066 19081 CAGCATTACATAAGAC 94 1485 915800 1270 1285 19110 19125 CTCTGGACAATCGCAA 55 1486 915820 1345 1360 23750 23765 CGAGTGAACACCTGTG 83 1487 915840 1402 1417 25165 25180 TGTTGGCTGCTCACTG 77 1488 915860 1470 1485 25233 25248 CTGGACAGCCCTTGGG 29 1489 915880 1539 1554 25302 25317 TATTGCCCAAGAAGAA 18 1490 915900 1598 1613 25361 25376 ACTCTTCTCTAGTGAA 67 1491 915920 1630 1645 25393 25408 CTGCTAGACTCGCCTC 88 1492 915940 1682 1697 25445 25460 AATGCGGAGGTAGCTG  0 1493 915960 1765 1780 25528 25543 AGGTTGCTTCCTAGCT 55 1494 915980 1785 1800 25548 25563 TGGACCGCTGCACAGG 82 1495 916000 1812 1827 25575 25590 GCATGCTGATGTATTA 52 1496 916020 1837 1852 25600 25615 TCATTTCCCAACCAGC 94 1497 916040 1881 1896 25644 25659 ACGAAACAGTCAGTAA 79 1498 916060 1907 1922 25670 25685 GGAACAGTCTGACCAT 22 1499 916080 1936 1951 25699 25714 AACACCTGTCATTCTA 71 1500 916100 1974 1989 25737 25752 GCCAGCCTACCCCCCA 23 1501 916120 2023 2038 25786 25801 AGTGGGATCATGCTAT  0 1502 916159 2136 2151 25899 25914 GTTACCCCCGCCATGG 47 1503 916179 2239 2254 26002 26017 TTCAACAGGTAACAAC 84 1504 916199 2621 2636 26384 26399 TGCACACTAGATTATT  0 1505 916219 2674 2689 26437 26452 GCGGAAGCTCCTGCTG  6 1506 916239 2704 2719 26467 26482 GTTCAAGTTGTGTGCT 85 1507 916259 2771 2786 26534 26549 CTCATCGCCACACATG 85 1508 916279 N/A N/A  4218  4233 CGGAATCTCATTTCCA  0 1509 916299 N/A N/A  4736  4751 GCAAATGATCATGTGG 93 1510 916319 N/A N/A  5396  5411 CTTAATGCTATCAGGT 83 1511 916339 N/A N/A  5649  5664 GGATATATTGGGCTCA 96 1512 916359 N/A N/A  6597  6612 AGGGTTTACAAACATG 32 1513 916379 N/A N/A  7466  7481 ATTCATTAGCATCACC 52 1514 916399 N/A N/A  7687  7702 GGTTAATCCATGGGTC  0 1515 916559 N/A N/A 12286 12301 AGTTTTAGGTCTGGGT 89 1516 916579 N/A N/A 12833 12848 CATTGCATAGCCTTCT 96 1517 916599 N/A N/A 13661 13676 CCATACATACCCTTCT 21 1518 916619 N/A N/A 14077 14092 ACCCACACCTGACTGG 16 1519 916639 N/A N/A 14572 14587 CGCTCCTACTTATCCC 96 1520 916659 N/A N/A 15427 15442 TCTTACAGCATTGGCA 38 1521 916679 N/A N/A 15973 15988 CATCTACCAAACTGCA 73 1522 916699 N/A N/A 17135 17150 AACAAACATCGATTTT 47 1523 916719 N/A N/A 17844 17859 AGCTTTACAAGCTGGT  0 1524 916739 N/A N/A 19213 19228 ACGACAATCCAGGTCC 14 1525 916778 N/A N/A 20612 20627 TCTAATTTTACGATCA 92 1526 916798 N/A N/A 20985 21000 ATTAAACTGCCAAGTC 72 1527 916818 N/A N/A 21441 21456 ACCAATATACTGAGAG 92 1528 916838 N/A N/A 22409 22424 AGCGGTAGCACCGCCA  0 1529 916858 N/A N/A 23323 23338 TCACATGTGAGCCCAG 46 1530 916878 N/A N/A 24271 24286 GTACAACAGAGGGTGG 19 1531 916898 N/A N/A 24978 24993 GGCTGATGTCACCACC 32 1532

TABLE 23 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915361   43   58  2781  2796 TCAGTGTCTCGGCCAG  0 1533 915381  105  120  2843  2858 TCGGCTCGGGTCCTGA  0 1534 915401  188  203  2926  2941 CAAGCTCCAGCCGCGC 22 1535 915421  247  262  2985  3000 CTCAGGCAGCGGGTCG 48 1536 915441  345  360  3083  3098 CCAGCGGGATACCGGA  0 1537 915461  384  399  5958  5973 CCTTCCGCACAAGATC 59 1538 915481  414  429  5988  6003 GATGGAAGATGCCAAT 83 1539 915501  450  465  6024  6039 GACCCTGTCGGAGGAA 26 1540 915521  482  497  6056  6071 CTGGTGGACATTGGCC  0 1541 915541  508  523  6082  6097 GAGATGCCTATTTTGC 92 1542 915561  557  572  6131  6146 CCGAAAGTCAGACACC  0 1543 915601  691  706 11909 11924 GCATCAATGAAGGGTA 91 1544 915621  751  766 11969 11984 TTGACTTTAGGGCAGA 85 1545 915641  822  837 12040 12055 GAAGGTAGAGGTTCCC 74 1546 915661  876  891 13612 13627 GAAGGCATATCTCTCC 32 1547 915681  930  945 16050 16065 GCCTGTTGCAGATGCC  0 1548 915701  983  998 16103 16118 CATGGCGACCTCAGGA  0 1549 915721 1035 1050 16155 16170 CCAAGGCAGCCGACTC 79 1550 915741 1122 1137 16242 16257 GCGAGAGGGTGTCCAG  0 1551 915761 1150 1165 18990 19005 TCTTCACTCAGTGCTG 41 1552 915781 1227 1242 19067 19082 GCAGCATTACATAAGA 75 1553 915801 1273 1288 N/A N/A AGTCTCTGGACAATCG  0 1554 915821 1346 1361 23751 23766 TCGAGTGAACACCTGT 52 1555 915841 1403 1418 25166 25181 CTGTTGGCTGCTCACT 80 1556 915861 1471 1486 25234 25249 GCTGGACAGCCCTTGG  0 1557 915881 1540 1555 25303 25318 TTATTGCCCAAGAAGA 75 1558 915901 1599 1614 25362 25377 GACTCTTCTCTAGTGA 67 1559 915921 1631 1646 25394 25409 TCTGCTAGACTCGCCT 50 1560 915941 1683 1698 25446 25461 CAATGCGGAGGTAGCT 39 1561 915961 1766 1781 25529 25544 AAGGTTGCTTCCTAGC 71 1562 915981 1786 1801 25549 25564 CTGGACCGCTGCACAG  0 1563 916001 1813 1828 25576 25591 CGCATGCTGATGTATT 73 1564 916021 1840 1855 25603 25618 GTGTCATTTCCCAACC 61 1565 916041 1882 1897 25645 25660 CACGAAACAGTCAGTA 34 1566 916061 1910 1925 25673 25688 GCTGGAACAGTCTGAC 82 1567 916081 1942 1957 25705 25720 CATCCAAACACCTGTC 69 1568 916101 1975 1990 25738 25753 GGCCAGCCTACCCCCC  2 1569 916121 2024 2039 25787 25802 AAGTGGGATCATGCTA 26 1570 916140 2095 2110 25858 25873 CATCTTTGCAGACCAC 92 1571 916160 2137 2152 25900 25915 TGTTACCCCCGCCATG 51 1572 916180 2257 2272 26020 26035 GATTCACATAATACAA 87 1573 916200 2622 2637 26385 26400 CTGCACACTAGATTAT  0 1574 916220 2675 2690 26438 26453 GGCGGAAGCTCCTGCT  0 1575 916240 2705 2720 26468 26483 GGTTCAAGTTGTGTGC 69 1576 916260 2772 2787 26535 26550 TCTCATCGCCACACAT 72 1577 916280 N/A N/A  4220  4235 TACGGAATCTCATTTC 80 1578 916300 N/A N/A  4791  4806 GGCCACCTTGGGATAC 17 1579 916320 N/A N/A  5398  5413 GCCTTAATGCTATCAG 67 1580 916340 N/A N/A  5650  5665 TGGATATATTGGGCTC 97 1581 916360 N/A N/A  6603  6618 ACATTCAGGGTTTACA 18 1582 916380 N/A N/A  7468  7483 GTATTCATTAGCATCA 51 1583 916400 N/A N/A  7688  7703 AGGTTAATCCATGGGT 29 1584 916560 N/A N/A 12287 12302 GAGTTTTAGGTCTGGG 95 1585 916580 N/A N/A 12905 12920 CTTATAAAGCACACGG 95 1586 916600 N/A N/A 13683 13698 GGGCATGGCTGATCCT  8 1587 916620 N/A N/A 14099 14114 CAAACTTGTCTAGTGG 67 1588 916640 N/A N/A 14600 14615 TCGCATCCATGGGTCC 83 1589 916660 N/A N/A 15429 15444 GCTCTTACAGCATTGG 51 1590 916680 N/A N/A 15974 15989 CCATCTACCAAACTGC 61 1591 916700 N/A N/A 17195 17210 GACTTAGTCCGTGTTC 49 1592 916720 N/A N/A 17883 17898 CAGCATCTATGTTCTC 67 1593 916740 N/A N/A 19239 19254 ATAGACTGTGAGCTGT 82 1594 916759 N/A N/A 20354 20369 GACCATTCTGCTCCCC 20 1595 916779 N/A N/A 20632 20647 GCCCATACCTTTTATC 47 1596 916799 N/A N/A 20987 21002 GTATTAAACTGCCAAG 81 1597 916819 N/A N/A 21444 21459 CTAACCAATATACTGA 73 1598 916839 N/A N/A 22506 22521 GGCTGGTGATGAAACA  0 1599 916859 N/A N/A 23345 23360 CCTCATGGTTTGCTGT 31 1600 916879 N/A N/A 24273 24288 CAGTACAACAGAGGGT 81 1601 916899 N/A N/A 25064 25079 CACATTGCCGGCCAGT 58 1602

TABLE 24 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915362   44   59  2782  2797 CTCAGTGTCTCGGCCA  0 1603 915382  106  121  2844  2859 ATCGGCTCGGGTCCTG 30 1604 915402  189  204  2927  2942 ACAAGCTCCAGCCGCG 24 1605 915422  248  263  2986  3001 GCTCAGGCAGCGGGTC 33 1606 915442  346  361 N/A N/A TCCAGCGGGATACCGG  0 1607 915462  385  400  5959  5974 GCCTTCCGCACAAGAT 60 1608 915482  415  430  5989  6004 GGATGGAAGATGCCAA 60 1609 915502  451  466  6025  6040 AGACCCTGTCGGAGGA 75 1610 915522  487  502  6061  6076 ATGAGCTGGTGGACAT 74 1611 915542  509  524  6083  6098 AGAGATGCCTATTTTG 91 1612 915562  558  573  6132  6147 ACCGAAAGTCAGACAC  0 1613 915602  692  707 11910 11925 GGCATCAATGAAGGGT 88 1614 915622  752  767 11970 11985 CTTGACTTTAGGGCAG 86 1615 915642  824  839 12042 12057 GAGAAGGTAGAGGTTC 81 1616 915662  878  893 13614 13629 TCGAAGGCATATCTCT 16 1617 915682  931  946 16051 16066 GGCCTGTTGCAGATGC  0 1618 915702  984  999 16104 16119 GCATGGCGACCTCAGG 24 1619 915722 1036 1051 16156 16171 GCCAAGGCAGCCGACT  0 1620 915742 1123 1138 16243 16258 GGCGAGAGGGTGTCCA  0 1621 915762 1173 1188 19013 19028 TGTATCCACCTTTGTC 85 1622 915782 1228 1243 19068 19083 GGCAGCATTACATAAG 73 1623 915802 1283 1298 N/A N/A CCATGTCACCAGTCTC 59 1624 915822 1347 1362 23752 23767 CTCGAGTGAACACCTG  0 1625 915842 1404 1419 25167 25182 CCTGTTGGCTGCTCAC 88 1626 915862 1472 1487 25235 25250 TGCTGGACAGCCCTTG  0 1627 915882 1541 1556 25304 25319 TTTATTGCCCAAGAAG 38 1628 915902 1600 1615 25363 25378 AGACTCTTCTCTAGTG 60 1629 915922 1632 1647 25395 25410 ATCTGCTAGACTCGCC 86 1630 915942 1684 1699 25447 25462 GCAATGCGGAGGTAGC 38 1631 915962 1767 1782 25530 25545 AAAGGTTGCTTCCTAG 77 1632 915982 1787 1802 25550 25565 GCTGGACCGCTGCACA 79 1633 916002 1814 1829 25577 25592 ACGCATGCTGATGTAT 72 1634 916022 1841 1856 25604 25619 GGTGTCATTTCCCAAC 80 1635 916042 1883 1898 25646 25661 CCACGAAACAGTCAGT 87 1636 916062 1912 1927 25675 25690 ATGCTGGAACAGTCTG 78 1637 916082 1943 1958 25706 25721 CCATCCAAACACCTGT 64 1638 916102 1976 1991 25739 25754 GGGCCAGCCTACCCCC  0 1639 916122 2025 2040 25788 25803 GAAGTGGGATCATGCT 71 1640 916141 2097 2112 25860 25875 ATCATCTTTGCAGACC 91 1641 916161 2138 2153 25901 25916 TTGTTACCCCCGCCAT 89 1642 916181 2259 2274 26022 26037 CTGATTCACATAATAC 91 1643 916201 2623 2638 26386 26401 CCTGCACACTAGATTA 59 1644 916221 2676 2691 26439 26454 AGGCGGAAGCTCCTGC  0 1645 916241 2706 2721 26469 26484 AGGTTCAAGTTGTGTG 87 1646 916281 N/A N/A  4224  4239 AATGTACGGAATCTCA 83 1647 916301 N/A N/A  4810  4825 GTCCATGTGGGTGTCC 74 1648 916321 N/A N/A  5399  5414 GGCCTTAATGCTATCA 13 1649 916341 N/A N/A  5711  5726 TAGTATGAAATATCTC 96 1650 916361 N/A N/A  6862  6877 ATTGTAACTGCCAGGC  0 1651 916381 N/A N/A  7471  7486 CCGGTATTCATTAGCA  0 1652 916401 N/A N/A  7728  7743 GAGCAGGGCAACAAAC 22 1653 916561 N/A N/A 12315 12330 ATATAACCACAGCCTG 54 1654 916581 N/A N/A 12906 12921 GCTTATAAAGCACACG 94 1655 916601 N/A N/A 13702 13717 TAGTAAATGCTTGTCA 95 1656 916621 N/A N/A 14123 14138 GGCAGAAATGTGCTCT 60 1657 916641 N/A N/A 14632 14647 CTTCATGCCATCCTGT 83 1658 916661 N/A N/A 15430 15445 TGCTCTTACAGCATTG  0 1659 916681 N/A N/A 16262 16277 GGTACCTGTAGCGAGC  0 1660 916701 N/A N/A 17197 17212 TTGACTTAGTCCGTGT 93 1661 916721 N/A N/A 18220 18235 AGCTACATCAGGCTGG  0 1662 916741 N/A N/A 19244 19259 TGCACATAGACTGTGA  0 1663 916760 N/A N/A 20373 20388 GACTGCTGAGCCAAGC 61 1664 916780 N/A N/A 20657 20672 AGAAATTGCAGTGCCC 92 1665 916800 N/A N/A 20988 21003 GGTATTAAACTGCCAA  0 1666 916820 N/A N/A 21447 21462 TCACTAACCAATATAC 47 1667 916840 N/A N/A 22602 22617 ATAAATCTGCAAGAGC 62 1668 916860 N/A N/A 23369 23384 TCTCATGGTCAAGACC 52 1669 916880 N/A N/A 24305 24320 GACTGCTAGGCTTCAC 54 1670 916900 N/A N/A 25100 25115 CGCTGCTGCAGTGTGC 34 1671

Human primer probe set RTS36075 (forward sequence TGAGGCTGGAGGGAGATG, designated herein as SEQ ID NO: 14; reverse sequence GCTCATGTATCCACCTTTGTCT, designated herein as SEQ ID NO: 15; probe sequence CTAGACCACCTGCGTCTCAGCATC, designated herein as SEQ ID NO: 16) was also used to measure mRNA levels. PNPLA3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of PNPLA3, relative to untreated control cells.

TABLE 25 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 898558  581  596 N/A N/A GGCATCCACGACTTCG 87 1672 912709   27   42  2765  2780 GGCATTCCCAGCGCGA  0   17 912710   95  110  2833  2848 TCCTGATCCGCAGCAG  0   18 912711  103  118  2841  2856 GGCTCGGGTCCTGATC  0   19 912712  131  146  2869  2884 GTTAGGATCTGGGTCG 76   20 912713  164  179  2902  2917 GTACATGGCGGCGGCG  0   21 912714  183  198  2921  2936 TCCAGCCGCGCTCTGC 29   22 912715  196  211  2934  2949 GCGAAGGACAAGCTCC 31   23 912716  197  212  2935  2950 CGCGAAGGACAAGCTC  0   24 912717  272  287  3010  3025 GCGGAGGAGGTGCGGG  0   25 912718  273  288  3011  3026 CGCGGAGGAGGTGCGG  0   26 912719  274  289  3012  3027 TCGCGGAGGAGGTGCG 16   27 912720  290  305  3028  3043 GAACAACATGCGCGCG  0   28 912721  291  306  3029  3044 CGAACAACATGCGCGC  2   29 912722  292  307  3030  3045 CCGAACAACATGCGCG  0   30 912723  293  308  3031  3046 GCCGAACAACATGCGC  0   31 912724  294  309  3032  3047 CGCCGAACAACATGCG  0   32 912725  323  338  3061  3076 GCCGACGCAGTGCAAC  0   33 912726  324  339  3062  3077 CGCCGACGCAGTGCAA  0   34 912727  340  355  3078  3093 GGGATACCGGAGAGGA 32   35 912728  370  385  5944  5959 TCTGAGAGGACCTGCA 31   36 912729  375  390  5949  5964 CAAGATCTGAGAGGAC 60   37 912730  404  419  5978  5993 GCCAATGTTCCGACTC 52   38 912731  410  425  5984  5999 GAAGATGCCAATGTTC 31   39 912732  429  444  6003  6018 TTAAGTTGAAGGATGG 93   40 912733  432  447  6006  6021 TGCTTAAGTTGAAGGA 82   41 912734  478  493  6052  6067 TGGACATTGGCCGGGA 73   42 912735  479  494  6053  6068 GTGGACATTGGCCGGG 44   43 912736  484  499  6058  6073 AGCTGGTGGACATTGG 29   44 912737  528  543  6102  6117 CATCAGACACTCTGGT  0   45 912738  531  546  6105  6120 CCCCATCAGACACTCT 55   46 912739  552  567  6126  6141 AGTCAGACACCAGAAC 23   47 912740  582  597 N/A N/A AGGCATCCACGACTTC 40 1673 912741  584  599 N/A N/A CAAGGCATCCACGACT 55 1674 912742  591  606 N/A N/A AACATACCAAGGCATC 59 1675 912743  593  608 N/A N/A GGAACATACCAAGGCA 69 1676 912744  594  609  7824  7839 AGGAACATACCAAGGC 85 1677 912745  625  640  7855  7870 GGGATAAGGCCACTGT 71 1678 912746  626  641  7856  7871 AGGGATAAGGCCACTG 12 1679 912747  630  645  7860  7875 AAGGAGGGATAAGGCC  0 1680 912748  652  667 N/A N/A ACATATCGCACGCCTC 35 1681 912749  653  668 N/A N/A CACATATCGCACGCCT  3 1682 912750  654  669 N/A N/A CCACATATCGCACGCC 27 1683 912751  656  671 N/A N/A ATCCACATATCGCACG 24 1684 912752  660  675 11878 11893 CTCCATCCACATATCG 87 1685 912753  689  704 11907 11922 ATCAATGAAGGGTACG 79 1686 912754  690  705 11908 11923 CATCAATGAAGGGTAC 63 1687 912755  693  708 11911 11926 TGGCATCAATGAAGGG 68   48 912756  698  713 11916 11931 TGTTTTGGCATCAATG 88   49 912757  746  761 11964 11979 TTTAGGGCAGATGTCG 75   50 912758  747  762 11965 11980 CTTTAGGGCAGATGTC 82   51 912759  795  810 12013 12028 GTAGACTGAGCTTGGT 96   52 912760  820  835 12038 12053 AGGTAGAGGTTCCCTG  0   53 912761  841  856 12059 12074 GGGACAAAAGCTCTCG  0   54 912762  873  888 13609 13624 GGCATATCTCTCCCAG  0   55 912763  874  889 13610 13625 AGGCATATCTCTCCCA  0   56 912764  886  901 13622 13637 AAATATCCTCGAAGGC 71   57 912765  888  903 13624 13639 CCAAATATCCTCGAAG 37   58 912766  889  904 13625 13640 TCCAAATATCCTCGAA  0   59 912767  894  909 13630 13645 ATGCATCCAAATATCC 42   60 912768  925  940 N/A N/A TTGCAGATGCCCTTCT  5   61 912769  968  983 16088 16103 ATCCATCCCTTCTGAG  6   62 912770  986 1001 16106 16121 GGGCATGGCGACCTCA  0   63 912771 1004 1019 16124 16139 ACTCATGTTTGCCCAG 67   64 912782 1195 1210 19035 19050 AGCAAGTTGCAAATCT 71   75 912783 1199 1214 19039 19054 GGGTAGCAAGTTGCAA 37   76 912784 1205 1220 19045 19060 CCTAATGGGTAGCAAG 25   77 912785 1206 1221 19046 19061 TCCTAATGGGTAGCAA 64   78

TABLE 26 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 912786 1207 1222 19047 19062 ATCCTAATGGGTAGCA 65  79 912787 1211 1226 19051 19066 CATTATCCTAATGGGT 43  80 912788 1212 1227 19052 19067 ACATTATCCTAATGGG  0  81 912789 1213 1228 19053 19068 GACATTATCCTAATGG 59  82 912790 1220 1235 19060 19075 TACATAAGACATTATC  8  83 912791 1224 1239 19064 19079 GCATTACATAAGACAT 86  84 912792 1245 1260 19085 19100 CCACAGGCAGGGTACA 58  85 912793 1246 1261 19086 19101 TCCACAGGCAGGGTAC  5  86 912794 1253 1268 19093 19108 GGCAGATTCCACAGGC 68  87 912795 1259 1274 19099 19114 CGCAATGGCAGATTCC 84  88 912796 1265 1280 19105 19120 GACAATCGCAATGGCA 63  89 912797 1266 1281 19106 19121 GGACAATCGCAATGGC 54  90 912798 1267 1282 19107 19122 TGGACAATCGCAATGG 59  91 912799 1285 1300 23690 23705 AGCCATGTCACCAGTC 51  92 912800 1289 1304 23694 23709 TGGAAGCCATGTCACC 32  93 912801 1290 1305 23695 23710 CTGGAAGCCATGTCAC 44  94 912802 1297 1312 23702 23717 GGCATATCTGGAAGCC  0  95 912803 1298 1313 23703 23718 GGGCATATCTGGAAGC  0  96 912804 1351 1366 23756 23771 AGCACTCGAGTGAACA  6  97 912805 1386 1401 N/A N/A GCATTTGGGACCTGGA 54  98 912806 1387 1402 N/A N/A GGCATTTGGGACCTGG 33  99 912807 1388 1403 25151 25166 TGGCATTTGGGACCTG  0 100 912808 1394 1409 25157 25172 GCTCACTGGCATTTGG  7 101 912809 1523 1538 25286 25301 GTTCAGGCTGGACCTG 17 102 912810 1547 1562 25310 25325 AGGTACTTTATTGCCC 30 103 912811 1550 1565 25313 25328 AGCAGGTACTTTATTG 55 104 912812 1653 1668 25416 25431 AACTTTAGCACCTCTG 87 105 912813 1655 1670 25418 25433 GAAACTTTAGCACCTC 85 106 912814 1656 1671 25419 25434 GGAAACTTTAGCACCT 26 107 912815 1669 1684 25432 25447 CTGCACAAAGATGGGA 66 108 912816 1671 1686 25434 25449 AGCTGCACAAAGATGG 41 109 912817 1685 1700 25448 25463 AGCAATGCGGAGGTAG 35 110 912818 1740 1755 25503 25518 ACCAACTCAGCTCAGA 76 111 912819 1741 1756 25504 25519 AACCAACTCAGCTCAG 77 112 912820 1757 1772 25520 25535 TCCTAGCTTTTCATAA 18 113 912821 1788 1803 25551 25566 TGCTGGACCGCTGCAC  1 114 912822 1796 1811 25559 25574 GAGTTAAGTGCTGGAC 90 115 912823 1802 1817 25565 25580 GTATTAGAGTTAAGTG 86 116 912824 1803 1818 25566 25581 TGTATTAGAGTTAAGT 79 117 912825 1806 1821 25569 25584 TGATGTATTAGAGTTA 89 118 912826 1808 1823 25571 25586 GCTGATGTATTAGAGT 79 119 912827 1821 1836 25584 25599 TGAATTAACGCATGCT 73 120 912828 1822 1837 25585 25600 CTGAATTAACGCATGC 69 121 912829 1870 1885 25633 25648 AGTAAGGGACCCTCTG  0 122 912830 1871 1886 25634 25649 CAGTAAGGGACCCTCT 44 123 912831 1872 1887 25635 25650 TCAGTAAGGGACCCTC 67 124 912832 1874 1889 25637 25652 AGTCAGTAAGGGACCC 50 125 912833 1893 1908 25656 25671 ATTAATAGGGCCACGA 78 126 912834 1895 1910 25658 25673 CCATTAATAGGGCCAC 72 127 912835 1896 1911 25659 25674 ACCATTAATAGGGCCA 65 128 912836 1906 1921 25669 25684 GAACAGTCTGACCATT 82 129 912837 1908 1923 25671 25686 TGGAACAGTCTGACCA 39 130 912838 1909 1924 25672 25687 CTGGAACAGTCTGACC 84 131 912839 1911 1926 25674 25689 TGCTGGAACAGTCTGA 72 132 912840 1916 1931 25679 25694 CCTCATGCTGGAACAG 84 133 912841 1928 1943 25691 25706 TCATTCTAAGAACCTC 87 134 912842 1945 1960 25708 25723 ACCCATCCAAACACCT 18 135 912843 1982 1997 25745 25760 ACACATGGGCCAGCCT 46 136 912844 1989 2004 25752 25767 CAAGATCACACATGGG 71 137 912845 2057 2072 25820 25835 GGGACGAACTGCACCC  0 138 912846 2098 2113 25861 25876 TATCATCTTTGCAGAC 68 139 912847 2116 2131 25879 25894 GTTTTTAGTAGTCAAG 90 140 912848 2117 2132 25880 25895 CGTTTTTAGTAGTCAA 94 141 912849 2145 2160 25908 25923 TATCATCTTGTTACCC 87 142 912850 2148 2163 25911 25926 GATTATCATCTTGTTA 60 143 912851 2150 2165 25913 25928 TAGATTATCATCTTGT 50 144 912852 2151 2166 25914 25929 GTAGATTATCATCTTG 72 145 912853 2152 2167 25915 25930 AGTAGATTATCATCTT 79 146 912854 2175 2190 25938 25953 GTGAAAAAGGTGTTCT 64 147 912855 2182 2197 25945 25960 TAGTTAGGTGAAAAAG 77 148 912856 2188 2203 25951 25966 TTATTTTAGTTAGGTG 82 149 912857 2190 2205 25953 25968 CATTATTTTAGTTAGG 77 150 912858 2273 2288 26036 26051 CTACTAACATCTCACT 48 151 912859 2274 2289 26037 26052 TCTACTAACATCTCAC 91 152 912860 2278 2293 26041 26056 TTATTCTACTAACATC 37 153 912861 2280 2295 26043 26058 GCTTATTCTACTAACA 77 154 912862 2281 2296 26044 26059 GGCTTATTCTACTAAC 70 155 912863 2632 2647 26395 26410 GGTGAATGCCCTGCAC 42 156 Study 2

Cultured A431 cells at a density of 5,000 cells per well were transfected by free uptake with 1,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PNPLA3 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS36070 was used to measure mRNA levels. PNPLA3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREENR. Results are presented as percent inhibition of PNPLA3, relative to untreated control cells.

TABLE 27 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938 TGATGGTTGTTTTGGC 97  702 959270  413  428  5987  6002 ATGGAAGATGCCAATG 32 1688 959280  491  506  6065  6080 GGAGATGAGCTGGTGG 66 1689 959290  793  808 12011 12026 AGACTGAGCTTGGTGA 78 1690 959300  899  914 13635 13650 CCTGAATGCATCCAAA 69 1691 959310 1084 1099 16204 16219 ATGCTGAGACGCAGGT  0 1692 959320 1256 1271 19096 19111 AATGGCAGATTCCACA 25 1693 959330 1642 1657 25405 25420 CTCTGAAAGAATCTGC 75 1694 959340 1659 1674 25422 25437 ATGGGAAACTTTAGCA 77 1695 959350 1839 1854 25602 25617 TGTCATTTCCCAACCA 79 1696 959360 2114 2129 25877 25892 TTTTAGTAGTCAAGGT 88 1697 959370 2223 2238 25986 26001 GCTTCCTTACATTTTT 85 1698 959380 2269 2284 26032 26047 TAACATCTCACTGATT 42 1699 959390 N/A N/A  4311  4326 CTAGTGAGAAACAAAC  0 1700 959400 N/A N/A  4761  4776 TTATTGTTGCTAAACC 32 1701 959410 N/A N/A  4863  4878 ACTTTAGGCTCCTGGG 60 1702 959420 N/A N/A  5285  5300 AGCCATAAATCTTGGG 24 1703 959430 N/A N/A  5573  5588 ATGACATCATGGCTTC 93 1704 959440 N/A N/A  5603  5618 TTATTCAATGTGGCTT 95 1705 959450 N/A N/A  5640  5655 GGGCTCAATGAAATTA 12 1706 959460 N/A N/A  5713  5728 CTTAGTATGAAATATC 86 1707 959470 N/A N/A  5808  5823 TACTGTCTACTATGGG 91 1708 959480 N/A N/A  6157  6172 CTTACATCCACGACTT 35 1709 959660 N/A N/A 12153 12168 CAGTAACTGGTAGCTC 74 1710 959670 N/A N/A 12169 12184 TGTTTGATTGTGCAGA 95 1711 959680 N/A N/A 12210 12225 CGCCTTTTATTTCCGT 92 1712 959690 N/A N/A 12313 12328 ATAACCACAGCCTGGG 66 1713 959700 N/A N/A 12675 12690 ATAAGAATCATCTTAG  7 1714 959710 N/A N/A 12711 12726 ACTACCGAACGCAGTT 41 1715 959720 N/A N/A 12757 12772 TAGAGTGGTAAGGCAT 84 1716 959730 N/A N/A 12793 12808 GGTTGGTGTACATGGT 96 1717 959740 N/A N/A 12880 12895 TCCTGTTAGACAGCTT 93 1718 959750 N/A N/A 12902 12917 ATAAAGCACACGGGAA 86 1719 959760 N/A N/A 12931 12946 TAAGAGCTGTCTCCTC 85 1720 959770 N/A N/A 12972 12987 CTAACAAACTTTGCAG 79 1721 959780 N/A N/A 13392 13407 TGTCACCCTTCCACGG 15 1722 959790 N/A N/A 13526 13541 ATTGGAAGACCGCAGA 43 1723 959800 N/A N/A 13706 13721 CCGCTAGTAAATGCTT 44 1724 959810 N/A N/A 13737 13752 AACTAAGGCAAATCTC 77 1725 959820 N/A N/A 13915 13930 GAGTCATGACATCCCA 89 1726 959830 N/A N/A 14299 14314 GCAGATAAATACACAT 93 1727 959840 N/A N/A 14424 14439 TTTCCCATCGACACAG 78 1728 959850 N/A N/A 14571 14586 GCTCCTACTTATCCCC 76 1729 959860 N/A N/A 15202 15217 TATTGCCAGGTATCTG 64 1730 959870 N/A N/A 15599 15614 CAATACATAGCAGAGC 23 1731 959880 N/A N/A 17192 17207 TTAGTCCGTGTTCAGG 90 1732 959890 N/A N/A 17222 17237 GTAGCTGGTTTGTGGG 20 1733 959900 N/A N/A 17295 17310 CATCTCTTAGGGCACC 79 1734 959910 N/A N/A 18393 18408 GTTTGGAAGTCGCCAT 77 1735 959920 N/A N/A 20284 20299 AGGAAGCCCAATCAAG 85 1736 959930 N/A N/A 20512 20527 CAGATTGAGTCTCCTG 10 1737 959940 N/A N/A 20607 20622 TTTTACGATCATCATT 72 1738 959950 N/A N/A 20661 20676 GCTTAGAAATTGCAGT 75 1739 959960 N/A N/A 20812 20827 AGGGTAATATTCAGAC 86 1740 959970 N/A N/A 20934 20949 TGTAGCAGACAGATCA 74 1741 959980 N/A N/A 21000 21015 TTTAACAGCTCAGGTA 66 1742 959990 N/A N/A 21405 21420 ATTCTAGACATGGCCA 51 1743 960000 N/A N/A 21442 21457 AACCAATATACTGAGA 71 1744 960010 N/A N/A 21545 21560 AGACATATGACATTTC 91 1745 960020 N/A N/A 22765 22780 ACATGACAGACTAACT 55 1746 960030 N/A N/A 24039 24054 CATCAATGCTGCACTC 13 1747

TABLE 28 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938 TGATGGTTGTTTTGGC 98  702 959271  425  440  5999  6014 GTTGAAGGATGGATGG 90 1748 959281  511  526  6085  6100 AGAGAGATGCCTATTT 73 1749 959291  813  828 12031 12046 GGTTCCCTGTGCAGAG 79 1750 959301  904  919 13640 13655 AAGAACCTGAATGCAT 48 1751 959311 1085 1100 16205 16220 GATGCTGAGACGCAGG  0 1752 959321 1602 1617 25365 25380 ACAGACTCTTCTCTAG 45 1753 959331 1643 1658 25406 25421 CCTCTGAAAGAATCTG 81 1754 959341 1673 1688 25436 25451 GTAGCTGCACAAAGAT 69 1755 959351 1842 1857 25605 25620 TGGTGTCATTTCCCAA 19 1756 959361 2115 2130 25878 25893 TTTTTAGTAGTCAAGG 91 1757 959371 2240 2255 26003 26018 ATTCAACAGGTAACAA 69 1758 959381 2271 2286 26034 26049 ACTAACATCTCACTGA 33 1759 959391 N/A N/A  4313  4328 AGCTAGTGAGAAACAA 47 1760 959401 N/A N/A  4764  4779 CTTTTATTGTTGCTAA 77 1761 959411 N/A N/A  4868  4883 AGTGTACTTTAGGCTC 90 1762 959421 N/A N/A  5286  5301 CAGCCATAAATCTTGG  0 1763 959431 N/A N/A  5574  5589 AATGACATCATGGCTT 74 1764 959441 N/A N/A  5604  5619 TTTATTCAATGTGGCT 96 1765 959451 N/A N/A  5642  5657 TTGGGCTCAATGAAAT  0 1766 959461 N/A N/A  5714  5729 GCTTAGTATGAAATAT 78 1767 959471 N/A N/A  5809  5824 GTACTGTCTACTATGG 78 1768 959481 N/A N/A  6160  6175 CTGCTTACATCCACGA  4 1769 959661 N/A N/A 12154 12169 ACAGTAACTGGTAGCT 72 1770 959671 N/A N/A 12170 12185 CTGTTTGATTGTGCAG 50 1771 959681 N/A N/A 12211 12226 GCGCCTTTTATTTCCG 14 1772 959691 N/A N/A 12322 12337 CCTGACTATATAACCA 56 1773 959701 N/A N/A 12689 12704 GACCGTGTTTCCAAAT 97 1774 959711 N/A N/A 12712 12727 AACTACCGAACGCAGT 48 1775 959721 N/A N/A 12759 12774 GGTAGAGTGGTAAGGC 95 1776 959731 N/A N/A 12828 12843 CATAGCCTTCTTTCTT 93 1777 959741 N/A N/A 12882 12897 AATCCTGTTAGACAGC 90 1778 959751 N/A N/A 12903 12918 TATAAAGCACACGGGA 82 1779 959761 N/A N/A 12933 12948 AATAAGAGCTGTCTCC 87 1780 959771 N/A N/A 12974 12989 CCCTAACAAACTTTGC 62 1781 959781 N/A N/A 13394 13409 AATGTCACCCTTCCAC 90 1782 959791 N/A N/A 13527 13542 CATTGGAAGACCGCAG 76 1783 959801 N/A N/A 13707 13722 ACCGCTAGTAAATGCT 35 1784 959811 N/A N/A 13740 13755 TAGAACTAAGGCAAAT 65 1785 959821 N/A N/A 13916 13931 GGAGTCATGACATCCC 42 1786 959831 N/A N/A 14302 14317 TGAGCAGATAAATACA 75 1787 959841 N/A N/A 14425 14440 CTTTCCCATCGACACA 79 1788 959851 N/A N/A 14573 14588 GCGCTCCTACTTATCC  0 1789 959861 N/A N/A 15203 15218 ATATTGCCAGGTATCT 67 1790 959871 N/A N/A 15763 15778 GTGTTGGTTTATAACA 13 1791 959881 N/A N/A 17194 17209 ACTTAGTCCGTGTTCA 45 1792 959891 N/A N/A 17224 17239 CTGTAGCTGGTTTGTG 42 1793 959901 N/A N/A 17296 17311 CCATCTCTTAGGGCAC 53 1794 959911 N/A N/A 18394 18409 TGTTTGGAAGTCGCCA 87 1795 959921 N/A N/A 20289 20304 ATCAGAGGAAGCCCAA 84 1796 959931 N/A N/A 20514 20529 ACCAGATTGAGTCTCC 91 1797 959941 N/A N/A 20613 20628 CTCTAATTTTACGATC 70 1798 959951 N/A N/A 20662 20677 AGCTTAGAAATTGCAG  0 1799 959961 N/A N/A 20813 20828 CAGGGTAATATTCAGA 87 1800 959971 N/A N/A 20936 20951 TTTGTAGCAGACAGAT 18 1801 959981 N/A N/A 21001 21016 TTTTAACAGCTCAGGT 71 1802 959991 N/A N/A 21406 21421 AATTCTAGACATGGCC 25 1803 960001 N/A N/A 21443 21458 TAACCAATATACTGAG 72 1804 960011 N/A N/A 22023 22038 CGCAAAAAGACAACGA 16 1805 960021 N/A N/A 22766 22781 GACATGACAGACTAAC 76 1806 960031 N/A N/A 24040 24055 CCATCAATGCTGCACT 61 1807

TABLE 29 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938 TGATGGTTGTTTTGGC 97  702 959272  426  441  6000  6015 AGTTGAAGGATGGATG 76 1808 959282  517  532  6091  6106 CTGGTAAGAGAGATGC 64 1809 959292  815  830 12033 12048 GAGGTTCCCTGTGCAG 81 1810 959302  905  920 13641 13656 CAAGAACCTGAATGCA 65 1811 959312 1087 1102 16207 16222 AGGATGCTGAGACGCA 66 1812 959322 1604 1619 25367 25382 TCACAGACTCTTCTCT 50 1813 959332 1644 1659 25407 25422 ACCTCTGAAAGAATCT 69 1814 959342 1675 1690 25438 25453 AGGTAGCTGCACAAAG 78 1815 959352 1903 1918 25666 25681 CAGTCTGACCATTAAT 77 1816 959362 2173 2188 25936 25951 GAAAAAGGTGTTCTAA 85 1817 959372 2242 2257 26005 26020 AAATTCAACAGGTAAC 62 1818 959382 2275 2290 26038 26053 TTCTACTAACATCTCA 80 1819 959392 N/A N/A  4732  4747 ATGATCATGTGGCGGT 80 1820 959402 N/A N/A  4765  4780 ACTTTTATTGTTGCTA 86 1821 959412 N/A N/A  4869  4884 GAGTGTACTTTAGGCT 94 1822 959422 N/A N/A  5389  5404 CTATCAGGTGCAGGAG 93 1823 959432 N/A N/A  5575  5590 CAATGACATCATGGCT 90 1824 959442 N/A N/A  5607  5622 TACTTTATTCAATGTG  0 1825 959452 N/A N/A  5643  5658 ATTGGGCTCAATGAAA 35 1826 959462 N/A N/A  5716  5731 TGGCTTAGTATGAAAT 80 1827 959472 N/A N/A  5864  5879 TTTGGCAAGGCCAGAA  0 1828 959482 N/A N/A  6960  6975 GCATAGAGGAAGCTCG 32 1829 959662 N/A N/A 12155 12170 GACAGTAACTGGTAGC 92 1830 959672 N/A N/A 12172 12187 TTCTGTTTGATTGTGC 97 1831 959682 N/A N/A 12280 12295 AGGTCTGGGTATATGT 93 1832 959692 N/A N/A 12323 12338 CCCTGACTATATAACC 32 1833 959702 N/A N/A 12691 12706 TTGACCGTGTTTCCAA 94 1834 959712 N/A N/A 12713 12728 AAACTACCGAACGCAG 92 1835 959722 N/A N/A 12760 12775 TGGTAGAGTGGTAAGG 94 1836 959732 N/A N/A 12829 12844 GCATAGCCTTCTTTCT 87 1837 959742 N/A N/A 12883 12898 CAATCCTGTTAGACAG 13 1838 959752 N/A N/A 12904 12919 TTATAAAGCACACGGG 87 1839 959762 N/A N/A 12934 12949 CAATAAGAGCTGTCTC 81 1840 959772 N/A N/A 13370 13385 TGCAGGCACCCCAGCA  0 1841 959782 N/A N/A 13395 13410 GAATGTCACCCTTCCA 90 1842 959792 N/A N/A 13528 13543 TCATTGGAAGACCGCA 84 1843 959802 N/A N/A 13708 13723 GACCGCTAGTAAATGC 57 1844 959812 N/A N/A 13741 13756 TTAGAACTAAGGCAAA 78 1845 959822 N/A N/A 13917 13932 TGGAGTCATGACATCC  0 1846 959832 N/A N/A 14303 14318 CTGAGCAGATAAATAC 74 1847 959842 N/A N/A 14553 14568 AGTCTTAATGTGGATT 76 1848 959852 N/A N/A 14667 14682 AGTGTCCCCATCCCCA 54 1849 959862 N/A N/A 15204 15219 AATATTGCCAGGTATC 56 1850 959872 N/A N/A 15765 15780 TAGTGTTGGTTTATAA 89 1851 959882 N/A N/A 17196 17211 TGACTTAGTCCGTGTT 43 1852 959892 N/A N/A 17225 17240 TCTGTAGCTGGTTTGT 61 1853 959902 N/A N/A 17298 17313 TCCCATCTCTTAGGGC 24 1854 959912 N/A N/A 18396 18411 TATGTTTGGAAGTCGC 91 1855 959922 N/A N/A 20290 20305 AATCAGAGGAAGCCCA 40 1856 959932 N/A N/A 20515 20530 AACCAGATTGAGTCTC 72 1857 959942 N/A N/A 20614 20629 TCTCTAATTTTACGAT 23 1858 959952 N/A N/A 20663 20678 CAGCTTAGAAATTGCA 24 1859 959962 N/A N/A 20814 20829 CCAGGGTAATATTCAG 87 1860 959972 N/A N/A 20937 20952 CTTTGTAGCAGACAGA 50 1861 959982 N/A N/A 21003 21018 TATTTTAACAGCTCAG 94 1862 959992 N/A N/A 21409 21424 TGCAATTCTAGACATG 12 1863 960002 N/A N/A 21445 21460 ACTAACCAATATACTG 55 1864 960012 N/A N/A 22541 22556 CAACAGATTACTGGAC 28 1865 960022 N/A N/A 22768 22783 AGGACATGACAGACTA 66 1866 960032 N/A N/A 24041 24056 ACCATCAATGCTGCAC 79 1867

TABLE 30 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938 TGATGGTTGTTTTGGC 98  702 959273  427  442  6001  6016 AAGTTGAAGGATGGAT 86 1868 959283  694  709 11912 11927 TTGGCATCAATGAAGG 73 1869 959293  816  831 12034 12049 AGAGGTTCCCTGTGCA 80 1870 959303  906  921 13642 13657 CCAAGAACCTGAATGC 65 1871 959313 1090 1105 16210 16225 GGCAGGATGCTGAGAC 43 1872 959323 1605 1620 25368 25383 CTCACAGACTCTTCTC 81 1873 959333 1646 1661 25409 25424 GCACCTCTGAAAGAAT 51 1874 959343 1677 1692 25440 25455 GGAGGTAGCTGCACAA 73 1875 959353 1904 1919 25667 25682 ACAGTCTGACCATTAA 85 1876 959363 2179 2194 25942 25957 TTAGGTGAAAAAGGTG 91 1877 959373 2258 2273 26021 26036 TGATTCACATAATACA 71 1878 959383 2277 2292 26040 26055 TATTCTACTAACATCT 38 1879 959393 N/A N/A  4733  4748 AATGATCATGTGGCGG 93 1880 959403 N/A N/A  4767  4782 TGACTTTTATTGTTGC 87 1881 959413 N/A N/A  4870  4885 TGAGTGTACTTTAGGC 94 1882 959423 N/A N/A  5392  5407 ATGCTATCAGGTGCAG  0 1883 959433 N/A N/A  5578  5593 GCACAATGACATCATG 86 1884 959443 N/A N/A  5608  5623 TTACTTTATTCAATGT  9 1885 959453 N/A N/A  5644  5659 TATTGGGCTCAATGAA 80 1886 959463 N/A N/A  5798  5813 TATGGGAGCCACATGT  4 1887 959473 N/A N/A  5866  5881 CTTTTGGCAAGGCCAG  0 1888 959483 N/A N/A  7199  7214 TTAAACAGAGGATGCA 31 1889 959663 N/A N/A 12156 12171 AGACAGTAACTGGTAG 75 1890 959673 N/A N/A 12199 12214 TCCGTTAACCATCAAG 95 1891 959683 N/A N/A 12282 12297 TTAGGTCTGGGTATAT 94 1892 959693 N/A N/A 12324 12339 CCCCTGACTATATAAC  0 1893 959703 N/A N/A 12692 12707 CTTGACCGTGTTTCCA 97 1894 959713 N/A N/A 12715 12730 TTAAACTACCGAACGC 95 1895 959723 N/A N/A 12761 12776 ATGGTAGAGTGGTAAG 77 1896 959733 N/A N/A 12832 12847 ATTGCATAGCCTTCTT 95 1897 959743 N/A N/A 12884 12899 CCAATCCTGTTAGACA 83 1898 959753 N/A N/A 12908 12923 CTGCTTATAAAGCACA  2 1899 959763 N/A N/A 12935 12950 ACAATAAGAGCTGTCT 85 1900 959773 N/A N/A 13372 13387 TTTGCAGGCACCCCAG 63 1901 959783 N/A N/A 13396 13411 TGAATGTCACCCTTCC 53 1902 959793 N/A N/A 13531 13546 GCATCATTGGAAGACC 86 1903 959803 N/A N/A 13713 13728 ACCAAGACCGCTAGTA 38 1904 959813 N/A N/A 13743 13758 TGTTAGAACTAAGGCA 79 1905 959823 N/A N/A 13919 13934 CCTGGAGTCATGACAT  7 1906 959833 N/A N/A 14304 14319 TCTGAGCAGATAAATA 39 1907 959843 N/A N/A 14554 14569 AAGTCTTAATGTGGAT 84 1908 959853 N/A N/A 14669 14684 TTAGTGTCCCCATCCC 78 1909 959863 N/A N/A 15205 15220 GAATATTGCCAGGTAT 86 1910 959873 N/A N/A 15766 15781 TTAGTGTTGGTTTATA 92 1911 959883 N/A N/A 17198 17213 TTTGACTTAGTCCGTG 86 1912 959893 N/A N/A 17226 17241 CTCTGTAGCTGGTTTG 82 1913 959903 N/A N/A 17601 17616 TTGATAGTGAATGTGT 83 1914 959913 N/A N/A 18397 18412 ATATGTTTGGAAGTCG 91 1915 959923 N/A N/A 20291 20306 CAATCAGAGGAAGCCC 34 1916 959933 N/A N/A 20516 20531 TAACCAGATTGAGTCT 66 1917 959943 N/A N/A 20615 20630 GTCTCTAATTTTACGA 53 1918 959953 N/A N/A 20664 20679 ACAGCTTAGAAATTGC 89 1919 959963 N/A N/A 20843 20858 CTGTATTAGCTCAATA 67 1920 959973 N/A N/A 20938 20953 TCTTTGTAGCAGACAG 84 1921 959983 N/A N/A 21004 21019 TTATTTTAACAGCTCA 92 1922 959993 N/A N/A 21410 21425 CTGCAATTCTAGACAT 29 1923 960003 N/A N/A 21535 21550 CATTTCAGAGTATAAG 46 1924 960013 N/A N/A 22708 22723 GATGTGAGTGAAATAA 69 1925 960023 N/A N/A 22769 22784 AAGGACATGACAGACT 68 1926 960033 N/A N/A 24043 24058 CCACCATCAATGCTGC 58 1927

TABLE 31 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938 TGATGGTTGTTTTGGC  99  702 959284  695  710 11913 11928 TTTGGCATCAATGAAG  69 1928 959294  817  832 12035 12050 TAGAGGTTCCCTGTGC  83 1929 959314 1091 1106 16211 16226 GGGCAGGATGCTGAGA  22 1930 959324 1606 1621 25369 25384 ACTCACAGACTCTTCT  68 1931 959334 1647 1662 25410 25425 AGCACCTCTGAAAGAA  67 1932 959344 1678 1693 25441 25456 CGGAGGTAGCTGCACA  85 1933 959354 1926 1941 25689 25704 ATTCTAAGAACCTCAT  51 1934 959384 N/A N/A  4303  4318 AAACAAACCCTCCGTC  10 1935 959394 N/A N/A  4734  4749 AAATGATCATGTGGCG  94 1936 959404 N/A N/A  4768  4783 CTGACTTTTATTGTTG  74 1937 959414 N/A N/A  4871  4886 GTGAGTGTACTTTAGG  98 1938 959424 N/A N/A  5393  5408 AATGCTATCAGGTGCA  31 1939 959434 N/A N/A  5579  5594 TGCACAATGACATCAT  88 1940 959444 N/A N/A  5621  5636 CTACCTGTGTCTTTTA  90 1941 959454 N/A N/A  5647  5662 ATATATTGGGCTCAAT  81 1942 959474 N/A N/A  5867  5882 ACTTTTGGCAAGGCCA  17 1943 959484 N/A N/A  7211  7226 CCGCAAACAAGGTTAA  22 1944 959684 N/A N/A 12283 12298 TTTAGGTCTGGGTATA  93 1945 959694 N/A N/A 12325 12340 CCCCCTGACTATATAA  18 1946 959704 N/A N/A 12693 12708 TCTTGACCGTGTTTCC  97 1947 959734 N/A N/A 12834 12849 GCATTGCATAGCCTTC  96 1948 959744 N/A N/A 12886 12901 AACCAATCCTGTTAGA  59 1949 959754 N/A N/A 12909 12924 TCTGCTTATAAAGCAC 100 1950 959774 N/A N/A 13373 13388 CTTTGCAGGCACCCCA  72 1951 959784 N/A N/A 13398 13413 CTTGAATGTCACCCTT  92 1952 959804 N/A N/A 13715 13730 TTACCAAGACCGCTAG  47 1953 959824 N/A N/A 14228 14243 ACTTTTAGTATTAAAG   0 1954 959844 N/A N/A 14555 14570 AAAGTCTTAATGTGGA  88 1955 959854 N/A N/A 14670 14685 CTTAGTGTCCCCATCC  76 1956 959874 N/A N/A 15767 15782 GTTAGTGTTGGTTTAT  95 1957 959884 N/A N/A 17199 17214 GTTTGACTTAGTCCGT  96 1958 959914 N/A N/A 18398 18413 AATATGTTTGGAAGTC  87 1959 959934 N/A N/A 20518 20533 AGTAACCAGATTGAGT  96 1960 959954 N/A N/A 20665 20680 CACAGCTTAGAAATTG  88 1961 959964 N/A N/A 20844 20859 CCTGTATTAGCTCAAT  92 1962 959974 N/A N/A 20940 20955 CCTCTTTGTAGCAGAC  84 1963 959984 N/A N/A 21006 21021 GGTTATTTTAACAGCT  85 1964 959994 N/A N/A 21412 21427 ACCTGCAATTCTAGAC  61 1965 960014 N/A N/A 22710 22725 AAGATGTGAGTGAAAT  70 1966 960024 N/A N/A 22770 22785 AAAGGACATGACAGAC  87 1967

TABLE 32 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 959275  440  455  6014  6029 GAGGAACTTGCTTAAG 81 1968 959285  696  711 11914 11929 TTTTGGCATCAATGAA 62 1969 959305 1066 1081 16186 16201 TCTAGCAGCTCATCTC 64 1970 959335 1649 1664 25412 25427 TTAGCACCTCTGAAAG 72 1971 959345 1804 1819 25567 25582 ATGTATTAGAGTTAAG 77 1972 959355 1927 1942 25690 25705 CATTCTAAGAACCTCA 71 1973 959365 2183 2198 25946 25961 TTAGTTAGGTGAAAAA 81 1974 959375 2261 2276 26024 26039 CACTGATTCACATAAT 70 1975 959395 N/A N/A  4737  4752 TGCAAATGATCATGTG 66 1976 959405 N/A N/A  4769  4784 GCTGACTTTTATTGTT 84 1977 959415 N/A N/A  4872  4887 AGTGAGTGTACTTTAG 94 1978 959425 N/A N/A  5395  5410 TTAATGCTATCAGGTG 81 1979 959435 N/A N/A  5580  5595 ATGCACAATGACATCA 86 1980 959445 N/A N/A  5624  5639 ATTCTACCTGTGTCTT 97 1981 959455 N/A N/A  5651  5666 TTGGATATATTGGGCT 97 1982 959475 N/A N/A  5868  5883 TACTTTTGGCAAGGCC 70 1983 959485 N/A N/A  7697  7712 GCACAGAGTAGGTTAA 72 1984 959655 N/A N/A 12146 12161 TGGTAGCTCCTGGCAA 55 1985 959675 N/A N/A 12201 12216 TTTCCGTTAACCATCA 94 1986 959695 N/A N/A 12667 12682 CATCTTAGTGGCTGGG 93 1987 959705 N/A N/A 12695 12710 GTTCTTGACCGTGTTT 97 1988 959715 N/A N/A 12717 12732 GGTTAAACTACCGAAC 11 1989 959725 N/A N/A 12783 12798 CATGGTCTGCAAATTT 89 1990 959745 N/A N/A 12887 12902 AAACCAATCCTGTTAG 46 1991 959755 N/A N/A 12910 12925 ATCTGCTTATAAAGCA 42 1992 959775 N/A N/A 13374 13389 ACTTTGCAGGCACCCC 87 1993 959785 N/A N/A 13399 13414 GCTTGAATGTCACCCT 94 1994 959805 N/A N/A 13716 13731 TTTACCAAGACCGCTA 86 1995 959825 N/A N/A 14230 14245 CAACTTTTAGTATTAA  0 1996 959835 N/A N/A 14417 14432 TCGACACAGCATCACC 62 1997 959855 N/A N/A 14671 14686 TCTTAGTGTCCCCATC 78 1998 959865 N/A N/A 15209 15224 TTAGGAATATTGCCAG 93 1999 959875 N/A N/A 15769 15784 GGGTTAGTGTTGGTTT 94 2000 959895 N/A N/A 17288 17303 TAGGGCACCTCAAGAA  0 2001 959915 N/A N/A 18400 18415 CAAATATGTTTGGAAG 50 2002 959925 N/A N/A 20293 20308 CCCAATCAGAGGAAGC 41 2003 959935 N/A N/A 20599 20614 TCATCATTATTACCTG 91 2004 959955 N/A N/A 20803 20818 TTCAGACCAGGGTAAT 91 2005 959965 N/A N/A 20845 20860 GCCTGTATTAGCTCAA 90 2006 959975 N/A N/A 20941 20956 GCCTCTTTGTAGCAGA  0 2007 959995 N/A N/A 21434 21449 TACTGAGAGGAAATGA 64 2008 960015 N/A N/A 22714 22729 TGTAAAGATGTGAGTG 78 2009 960025 N/A N/A 22772 22787 TCAAAGGACATGACAG 80 2010 960037 N/A N/A 22590 22605 GAGCAACGAGGAAGGA 68 2011

TABLE 33 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938 TGATGGTTGTTTTGGC 98  702 959276  447  462  6021  6036 CCTGTCGGAGGAACTT 37 2012 959286  699  714 11917 11932 TTGTTTTGGCATCAAT 73 2013 959296  895  910 13631 13646 AATGCATCCAAATATC  0 2014 959306 1069 1084 16189 16204 TGGTCTAGCAGCTCAT 25 2015 959316 1221 1236 19061 19076 TTACATAAGACATTAT 35 2016 959326 1614 1629 25377 25392 CTCAAGTGACTCACAG 85 2017 959336 1650 1665 25413 25428 TTTAGCACCTCTGAAA 24 2018 959346 1834 1849 25597 25612 TTTCCCAACCAGCTGA 60 2019 959356 2096 2111 25859 25874 TCATCTTTGCAGACCA 90 2020 959366 2184 2199 25947 25962 TTTAGTTAGGTGAAAA 57 2021 959376 2262 2277 26025 26040 TCACTGATTCACATAA 86 2022 959386 N/A N/A  4306  4321 GAGAAACAAACCCTCC  0 2023 959396 N/A N/A  4738  4753 GTGCAAATGATCATGT 69 2024 959406 N/A N/A  4771  4786 AAGCTGACTTTTATTG 57 2025 959416 N/A N/A  5276  5291 TCTTGGGATGCACAGG 49 2026 959426 N/A N/A  5397  5412 CCTTAATGCTATCAGG  0 2027 959436 N/A N/A  5581  5596 AATGCACAATGACATC 69 2028 959446 N/A N/A  5625  5640 AATTCTACCTGTGTCT 82 2029 959456 N/A N/A  5652  5667 TTTGGATATATTGGGC 95 2030 959466 N/A N/A  5802  5817 CTACTATGGGAGCCAC 64 2031 959476 N/A N/A  5871  5886 TAATACTTTTGGCAAG 29 2032 959486 N/A N/A  7784  7799 TTATAGGCGAGAGCAC  0 2033 959656 N/A N/A 12147 12162 CTGGTAGCTCCTGGCA 44 2034 959666 N/A N/A 12164 12179 GATTGTGCAGACAGTA 95 2035 959676 N/A N/A 12202 12217 ATTTCCGTTAACCATC 93 2036 959686 N/A N/A 12288 12303 TGAGTTTTAGGTCTGG 96 2037 959696 N/A N/A 12669 12684 ATCATCTTAGTGGCTG 91 2038 959706 N/A N/A 12696 12711 TGTTCTTGACCGTGTT 98 2039 959716 N/A N/A 12719 12734 AAGGTTAAACTACCGA  6 2040 959726 N/A N/A 12785 12800 TACATGGTCTGCAAAT 90 2041 959736 N/A N/A 12838 12853 CATTGCATTGCATAGC 96 2042 959746 N/A N/A 12888 12903 AAAACCAATCCTGTTA 47 2043 959756 N/A N/A 12911 12926 CATCTGCTTATAAAGC 81 2044 959766 N/A N/A 12967 12982 AAACTTTGCAGCCTAT 93 2045 959776 N/A N/A 13376 13391 AGACTTTGCAGGCACC 90 2046 959786 N/A N/A 13400 13415 GGCTTGAATGTCACCC 69 2047 959796 N/A N/A 13697 13712 AATGCTTGTCAAAAGG 70 2048 959806 N/A N/A 13717 13732 CTTTACCAAGACCGCT 82 2049 959816 N/A N/A 13747 13762 TAAGTGTTAGAACTAA 30 2050 959826 N/A N/A 14232 14247 ACCAACTTTTAGTATT 79 2051 959836 N/A N/A 14419 14434 CATCGACACAGCATCA 59 2052 959846 N/A N/A 14557 14572 CCAAAGTCTTAATGTG 53 2053 959856 N/A N/A 14676 14691 CCATCTCTTAGTGTCC 88 2054 959866 N/A N/A 15210 15225 CTTAGGAATATTGCCA 87 2055 959876 N/A N/A 15770 15785 AGGGTTAGTGTTGGTT 89 2056 959886 N/A N/A 17202 17217 TCTGTTTGACTTAGTC 70 2057 959896 N/A N/A 17290 17305 CTTAGGGCACCTCAAG 30 2058 959906 N/A N/A 17735 17750 TAATCTGGTCATATGG 43 2059 959916 N/A N/A 18445 18460 TGCTTACGGAGCATAG  0 2060 959926 N/A N/A 20472 20487 CTCTAGACGGGAAGCT 31 2061 959936 N/A N/A 20601 20616 GATCATCATTATTACC 71 2062 959946 N/A N/A 20652 20667 TTGCAGTGCCCTGGCC 31 2063 959956 N/A N/A 20804 20819 ATTCAGACCAGGGTAA 87 2064 959966 N/A N/A 20847 20862 ATGCCTGTATTAGCTC 65 2065 959976 N/A N/A 20942 20957 AGCCTCTTTGTAGCAG  5 2066 959986 N/A N/A 21008 21023 GAGGTTATTTTAACAG 69 2067 959996 N/A N/A 21435 21450 ATACTGAGAGGAAATG 68 2068 960006 N/A N/A 21539 21554 ATGACATTTCAGAGTA 88 2069 960016 N/A N/A 22715 22730 GTGTAAAGATGTGAGT 84 2070 960026 N/A N/A 24033 24048 TGCTGCACTCAAAGAG  0 2071 960038 N/A N/A 19377 19392 TCACAAGAGACTGGAC 31 2072

TABLE 34 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938 TGATGGTTGTTTTGGC 98  702 959277  481  496  6055  6070 TGGTGGACATTGGCCG  5 2073 959287  707  722 11925 11940 GGTGATGGTTGTTTTG 85 2074 959297  896  911 13632 13647 GAATGCATCCAAATAT  0 2075 959307 1070 1085 16190 16205 GTGGTCTAGCAGCTCA 66 2076 959317 1223 1238 19063 19078 CATTACATAAGACATT 46 2077 959327 1615 1630 25378 25393 CCTCAAGTGACTCACA 83 2078 959337 1651 1666 25414 25429 CTTTAGCACCTCTGAA 71 2079 959347 1835 1850 25598 25613 ATTTCCCAACCAGCTG 49 2080 959357 2099 2114 25862 25877 TTATCATCTTTGCAGA 48 2081 959367 2185 2200 25948 25963 TTTTAGTTAGGTGAAA 43 2082 959377 2263 2278 26026 26041 CTCACTGATTCACATA 79 2083 959387 N/A N/A  4307  4322 TGAGAAACAAACCCTC  0 2084 959397 N/A N/A  4739  4754 TGTGCAAATGATCATG 82 2085 959407 N/A N/A  4859  4874 TAGGCTCCTGGGACCT  0 2086 959417 N/A N/A  5277  5292 ATCTTGGGATGCACAG 89 2087 959427 N/A N/A  5567  5582 TCATGGCTTCCAGTGT 78 2088 959437 N/A N/A  5600  5615 TTCAATGTGGCTTCTA 96 2089 959447 N/A N/A  5627  5642 TTAATTCTACCTGTGT 72 2090 959457 N/A N/A  5706  5721 TGAAATATCTCATTAG 77 2091 959467 N/A N/A  5805  5820 TGTCTACTATGGGAGC 83 2092 959477 N/A N/A  5875  5890 ATGGTAATACTTTTGG 75 2093 959657 N/A N/A 12148 12163 ACTGGTAGCTCCTGGC 78 2094 959667 N/A N/A 12165 12180 TGATTGTGCAGACAGT 97 2095 959677 N/A N/A 12203 12218 TATTTCCGTTAACCAT 91 2096 959687 N/A N/A 12289 12304 CTGAGTTTTAGGTCTG 96 2097 959697 N/A N/A 12671 12686 GAATCATCTTAGTGGC 94 2098 959707 N/A N/A 12697 12712 TTGTTCTTGACCGTGT 98 2099 959717 N/A N/A 12753 12768 GTGGTAAGGCATACTA 35 2100 959727 N/A N/A 12789 12804 GGTGTACATGGTCTGC 97 2101 959737 N/A N/A 12839 12854 GCATTGCATTGCATAG 92 2102 959747 N/A N/A 12890 12905 GGAAAACCAATCCTGT 69 2103 959757 N/A N/A 12927 12942 AGCTGTCTCCTCTACT 70 2104 959767 N/A N/A 12968 12983 CAAACTTTGCAGCCTA 95 2105 959777 N/A N/A 13377 13392 GAGACTTTGCAGGCAC 88 2106 959787 N/A N/A 13402 13417 TCGGCTTGAATGTCAC 67 2107 959797 N/A N/A 13700 13715 GTAAATGCTTGTCAAA 91 2108 959807 N/A N/A 13720 13735 AGTCTTTACCAAGACC  0 2109 959817 N/A N/A 13911 13926 CATGACATCCCAGTTC 29 2110 959827 N/A N/A 14233 14248 AACCAACTTTTAGTAT 27 2111 959837 N/A N/A 14420 14435 CCATCGACACAGCATC 89 2112 959847 N/A N/A 14567 14582 CTACTTATCCCCAAAG 16 2113 959857 N/A N/A 14677 14692 GCCATCTCTTAGTGTC 36 2114 959867 N/A N/A 15211 15226 CCTTAGGAATATTGCC 75 2115 959877 N/A N/A 15771 15786 GAGGGTTAGTGTTGGT 93 2116 959887 N/A N/A 17218 17233 CTGGTTTGTGGGTTCT 75 2117 959897 N/A N/A 17291 17306 TCTTAGGGCACCTCAA 53 2118 959907 N/A N/A 17736 17751 TTAATCTGGTCATATG  0 2119 959917 N/A N/A 18852 18867 ACAAAAGCGACAAGGT 33 2120 959927 N/A N/A 20508 20523 TTGAGTCTCCTGACCA 65 2121 959937 N/A N/A 20602 20617 CGATCATCATTATTAC 87 2122 959947 N/A N/A 20653 20668 ATTGCAGTGCCCTGGC 69 2123 959957 N/A N/A 20805 20820 TATTCAGACCAGGGTA 89 2124 959967 N/A N/A 20848 20863 GATGCCTGTATTAGCT 72 2125 959977 N/A N/A 20944 20959 GCAGCCTCTTTGTAGC  0 2126 959987 N/A N/A 21010 21025 CTGAGGTTATTTTAAC 40 2127 959997 N/A N/A 21436 21451 TATACTGAGAGGAAAT 47 2128 960007 N/A N/A 21541 21556 ATATGACATTTCAGAG 91 2129 960017 N/A N/A 22716 22731 CGTGTAAAGATGTGAG 87 2130 960027 N/A N/A 24035 24050 AATGCTGCACTCAAAG 31 2131 960039 N/A N/A 20215 20230 TAACAAACTATGCCTA 44 2132

TABLE 35 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938 TGATGGTTGTTTTGGC 98  702 959274  439  454  6013  6028 AGGAACTTGCTTAAGT 73 2133 959284  695  710 11913 11928 TTTGGCATCAATGAAG 60 1928 959294  817  832 12035 12050 TAGAGGTTCCCTGTGC 77 1929 959304 1063 1078 16183 16198 AGCAGCTCATCTCCCT 62 2134 959314 1091 1106 16211 16226 GGGCAGGATGCTGAGA 13 1930 959324 1606 1621 25369 25384 ACTCACAGACTCTTCT 72 1931 959334 1647 1662 25410 25425 AGCACCTCTGAAAGAA 65 1932 959344 1678 1693 25441 25456 CGGAGGTAGCTGCACA 86 1933 959354 1926 1941 25689 25704 ATTCTAAGAACCTCAT 54 1934 959364 2181 2196 25944 25959 AGTTAGGTGAAAAAGG 92 2135 959374 2260 2275 26023 26038 ACTGATTCACATAATA 78 2136 959384 N/A N/A  4303  4318 AAACAAACCCTCCGTC  2 1935 959394 N/A N/A  4734  4749 AAATGATCATGTGGCG 94 1936 959404 N/A N/A  4768  4783 CTGACTTTTATTGTTG 72 1937 959414 N/A N/A  4871  4886 GTGAGTGTACTTTAGG 97 1938 959424 N/A N/A  5393  5408 AATGCTATCAGGTGCA 31 1939 959434 N/A N/A  5579  5594 TGCACAATGACATCAT 87 1940 959444 N/A N/A  5621  5636 CTACCTGTGTCTTTTA 90 1941 959454 N/A N/A  5647  5662 ATATATTGGGCTCAAT 80 1942 959464 N/A N/A  5799  5814 CTATGGGAGCCACATG 24 2137 959474 N/A N/A  5867  5882 ACTTTTGGCAAGGCCA 23 1943 959484 N/A N/A  7211  7226 CCGCAAACAAGGTTAA  0 1944 959664 N/A N/A 12157 12172 CAGACAGTAACTGGTA 96 2138 959674 N/A N/A 12200 12215 TTCCGTTAACCATCAA 97 2139 959684 N/A N/A 12283 12298 TTTAGGTCTGGGTATA 93 1945 959694 N/A N/A 12325 12340 CCCCCTGACTATATAA 26 1946 959704 N/A N/A 12693 12708 TCTTGACCGTGTTTCC 98 1947 959714 N/A N/A 12716 12731 GTTAAACTACCGAACG 35 2140 959724 N/A N/A 12763 12778 CTATGGTAGAGTGGTA 93 2141 959734 N/A N/A 12834 12849 GCATTGCATAGCCTTC 97 1948 959744 N/A N/A 12886 12901 AACCAATCCTGTTAGA 55 1949 959754 N/A N/A 12909 12924 TCTGCTTATAAAGCAC  0 1950 959764 N/A N/A 12937 12952 GGACAATAAGAGCTGT 91 2142 959774 N/A N/A 13373 13388 CTTTGCAGGCACCCCA 72 1951 959784 N/A N/A 13398 13413 CTTGAATGTCACCCTT 92 1952 959794 N/A N/A 13532 13547 AGCATCATTGGAAGAC 92 2143 959804 N/A N/A 13715 13730 TTACCAAGACCGCTAG 57 1953 959814 N/A N/A 13744 13759 GTGTTAGAACTAAGGC 94 2144 959824 N/A N/A 14228 14243 ACTTTTAGTATTAAAG  0 1954 959834 N/A N/A 14306 14321 TTTCTGAGCAGATAAA 66 2145 959844 N/A N/A 14555 14570 AAAGTCTTAATGTGGA 87 1955 959854 N/A N/A 14670 14685 CTTAGTGTCCCCATCC 77 1956 959864 N/A N/A 15208 15223 TAGGAATATTGCCAGG 89 2146 959874 N/A N/A 15767 15782 GTTAGTGTTGGTTTAT 94 1957 959884 N/A N/A 17199 17214 GTTTGACTTAGTCCGT 95 1958 959894 N/A N/A 17228 17243 AACTCTGTAGCTGGTT 41 2147 959904 N/A N/A 17603 17618 TCTTGATAGTGAATGT 73 2148 959914 N/A N/A 18398 18413 AATATGTTTGGAAGTC 89 1959 959924 N/A N/A 20292 20307 CCAATCAGAGGAAGCC 58 2149 959934 N/A N/A 20518 20533 AGTAACCAGATTGAGT 81 1960 959944 N/A N/A 20617 20632 CTGTCTCTAATTTTAC 75 2150 959954 N/A N/A 20665 20680 CACAGCTTAGAAATTG 87 1961 959964 N/A N/A 20844 20859 CCTGTATTAGCTCAAT 92 1962 959974 N/A N/A 20940 20955 CCTCTTTGTAGCAGAC 83 1963 959984 N/A N/A 21006 21021 GGTTATTTTAACAGCT 85 1964 959994 N/A N/A 21412 21427 ACCTGCAATTCTAGAC 54 1965 960004 N/A N/A 21537 21552 GACATTTCAGAGTATA 93 2151 960014 N/A N/A 22710 22725 AAGATGTGAGTGAAAT 69 1966 960024 N/A N/A 22770 22785 AAAGGACATGACAGAC 75 1967 960034 N/A N/A 24613 24628 GCAAATCGGATCTTTG 32 2152

TABLE 36 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site Site Site Sequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938 TGATGGTTGTTTTGGC 98  702 959275  440  455  6014  6029 GAGGAACTTGCTTAAG 80 1968 959285  696  711 11914 11929 TTTTGGCATCAATGAA 63 1969 959295  823  838 12041 12056 AGAAGGTAGAGGTTCC 48 2153 959305 1066 1081 16186 16201 TCTAGCAGCTCATCTC 66 1970 959315 1093 1108 16213 16228 CAGGGCAGGATGCTGA  2 2154 959325 1608 1623 25371 25386 TGACTCACAGACTCTT 60 2155 959335 1649 1664 25412 25427 TTAGCACCTCTGAAAG 54 1971 959345 1804 1819 25567 25582 ATGTATTAGAGTTAAG 79 1972 959355 1927 1942 25690 25705 CATTCTAAGAACCTCA 68 1973 959365 2183 2198 25946 25961 TTAGTTAGGTGAAAAA 69 1974 959375 2261 2276 26024 26039 CACTGATTCACATAAT 73 1975 959385 N/A N/A  4305  4320 AGAAACAAACCCTCCG 70 2156 959395 N/A N/A  4737  4752 TGCAAATGATCATGTG 69 1976 959405 N/A N/A  4769  4784 GCTGACTTTTATTGTT 83 1977 959415 N/A N/A  4872  4887 AGTGAGTGTACTTTAG 94 1978 959425 N/A N/A  5395  5410 TTAATGCTATCAGGTG 82 1979 959435 N/A N/A  5580  5595 ATGCACAATGACATCA 84 1980 959445 N/A N/A  5624  5639 ATTCTACCTGTGTCTT 95 1981 959455 N/A N/A  5651  5666 TTGGATATATTGGGCT 97 1982 959465 N/A N/A  5800  5815 ACTATGGGAGCCACAT 26 2157 959475 N/A N/A  5868  5883 TACTTTTGGCAAGGCC 69 1983 959485 N/A N/A  7697  7712 GCACAGAGTAGGTTAA 70 1984 959655 N/A N/A 12146 12161 TGGTAGCTCCTGGCAA 50 1985 959665 N/A N/A 12162 12177 TTGTGCAGACAGTAAC 87 2158 959675 N/A N/A 12201 12216 TTTCCGTTAACCATCA 95 1986 959685 N/A N/A 12284 12299 TTTTAGGTCTGGGTAT 81 2159 959695 N/A N/A 12667 12682 CATCTTAGTGGCTGGG 91 1987 959705 N/A N/A 12695 12710 GTTCTTGACCGTGTTT 97 1988 959715 N/A N/A 12717 12732 GGTTAAACTACCGAAC 26 1989 959725 N/A N/A 12783 12798 CATGGTCTGCAAATTT 89 1990 959735 N/A N/A 12837 12852 ATTGCATTGCATAGCC 95 2160 959745 N/A N/A 12887 12902 AAACCAATCCTGTTAG 54 1991 959755 N/A N/A 12910 12925 ATCTGCTTATAAAGCA 43 1992 959765 N/A N/A 12964 12979 CTTTGCAGCCTATCCC 95 2161 959775 N/A N/A 13374 13389 ACTTTGCAGGCACCCC 86 1993 959785 N/A N/A 13399 13414 GCTTGAATGTCACCCT 95 1994 959795 N/A N/A 13534 13549 TCAGCATCATTGGAAG 60 2162 959805 N/A N/A 13716 13731 TTTACCAAGACCGCTA 82 1995 959815 N/A N/A 13745 13760 AGTGTTAGAACTAAGG 93 2163 959825 N/A N/A 14230 14245 CAACTTTTAGTATTAA  9 1996 959835 N/A N/A 14417 14432 TCGACACAGCATCACC 59 1997 959845 N/A N/A 14556 14571 CAAAGTCTTAATGTGG 84 2164 959855 N/A N/A 14671 14686 TCTTAGTGTCCCCATC 78 1998 959865 N/A N/A 15209 15224 TTAGGAATATTGCCAG 91 1999 959875 N/A N/A 15769 15784 GGGTTAGTGTTGGTTT 93 2000 959885 N/A N/A 17200 17215 TGTTTGACTTAGTCCG 96 2165 959895 N/A N/A 17288 17303 TAGGGCACCTCAAGAA  0 2001 959905 N/A N/A 17734 17749 AATCTGGTCATATGGT 42 2166 959915 N/A N/A 18400 18415 CAAATATGTTTGGAAG 55 2002 959925 N/A N/A 20293 20308 CCCAATCAGAGGAAGC 57 2003 959935 N/A N/A 20599 20614 TCATCATTATTACCTG 93 2004 959945 N/A N/A 20651 20666 TGCAGTGCCCTGGCCT 40 2167 959955 N/A N/A 20803 20818 TTCAGACCAGGGTAAT 93 2005 959965 N/A N/A 20845 20860 GCCTGTATTAGCTCAA 88 2006 959975 N/A N/A 20941 20956 GCCTCTTTGTAGCAGA  0 2007 959985 N/A N/A 21007 21022 AGGTTATTTTAACAGC 94 2168 959995 N/A N/A 21434 21449 TACTGAGAGGAAATGA 65 2008 960005 N/A N/A 21538 21553 TGACATTTCAGAGTAT 87 2169 960015 N/A N/A 22714 22729 TGTAAAGATGTGAGTG 75 2009 960025 N/A N/A 22772 22787 TCAAAGGACATGACAG 78 2010 960037 N/A N/A 22590 22605 GAGCAACGAGGAAGGA 64 2011

Example 2: Dose-Dependent Antisense Inhibition of Human PNPLA3 in A431 Cells

Gapmers from Example 1 exhibiting significant in vitro inhibition of PNPLA3 mRNA were selected and tested at various doses in A431 cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 10,000 cells per well and transfected free uptake with different concentrations of antisense oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and PNPLA3 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS36070 was used to measure mRNA levels. PNPLA3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of PNPLA3, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. PNPLA3 mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 37 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM) 912712 27 67 76 74 0.2 912732 54 78 88 87 <0.1 912733 45 74 85 88 <0.1 912734 33 64 80 83 0.1 912756 46 72 89 92 <0.1 912757 31 62 78 86 0.2 912758 38 70 85 90 0.1 912759 66 92 97 98 <0.1 912772 46 63 79 88 0.1 912795 40 64 83 84 0.1 912812 43 81 88 88 <0.1 912822 81 83 92 86 <0.1 912823 67 80 91 86 <0.1 912825 58 80 86 88 <0.1 912834 37 75 81 84 0.1 912841 17 62 79 69 0.3 912847 70 83 90 91 <0.1 912848 80 89 90 90 <0.1 912855 48 62 77 80 0.1

TABLE 38 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM) 912759 68 94 94 98 <0.1 912813 57 84 90 87 <0.1 912856 60 81 91 88 <0.1 912859 48 79 81 72 <0.1 912864 60 88 90 90 <0.1 912870 67 81 91 94 <0.1 912871 21 67 84 89 0.2 912872 18 73 90 92 0.2 912876 43 70 87 92 0.1 912933 68 89 90 90 <0.1 912940 86 91 95 96 <0.1 912941 87 94 96 96 <0.1 912952 68 85 90 91 <0.1 912953 80 90 95 93 <0.1 912964 59 78 88 91 <0.1 912973 53 70 87 91 <0.1 912980 54 77 84 88 <0.1 912985 23 61 81 87 0.2 912988 65 83 86 89 <0.1

TABLE 39 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM) 912759 72 95 97 99 <0.1 912874 78 90 96 97 <0.1 912875 64 83 92 94 <0.1 912886 49 78 85 92 <0.1 912931 68 88 94 95 <0.1 912934 57 83 90 92 <0.1 912936 50 78 89 89 <0.1 912938 57 73 85 87 <0.1 912943 64 84 90 93 <0.1 912954 80 92 93 94 <0.1 912970 44 73 86 90 <0.1 912986 56 78 91 92 <0.1 912987 79 90 92 88 <0.1 912992 21 59 74 81 0.3 915603 50 88 96 98 <0.1 915623 81 96 98 98 <0.1 915643 67 89 94 96 <0.1 916602 79 92 95 96 <0.1 916642 44 83 91 93 <0.1

TABLE 40 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM) 912759 73 94 98 99 <0.1 915484 67 87 93 95 <0.1 915543 34 69 87 90 0.1 915604 54 78 91 95 <0.1 915763 63 80 87 87 <0.1 915904 50 83 92 94 <0.1 915923 63 74 82 87 <0.1 916183 33 78 89 91 0.1 916303 58 73 84 91 <0.1 916343 15 72 76 87 0.2 916563 46 74 90 95 <0.1 916582 48 74 89 91 <0.1 916623 64 81 91 94 <0.1 916702 45 70 78 79 <0.1 916761 46 75 85 88 <0.1 916781 55 79 86 87 <0.1 916782 62 87 91 93 <0.1 916802 66 88 94 91 <0.1 916822 29 72 83 87 0.1

TABLE 41 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM) 912759 72 95 98 99 <0.1 915525 51 76 88 84 <0.1 915546 39 79 90 94 0.1 915605 59 84 96 96 <0.1 915606 74 94 99 98 <0.1 915625 72 82 91 95 <0.1 915944 36 71 75 83 0.1 916065 36 62 78 79 0.1 916144 71 86 90 92 <0.1 916163 36 67 81 74 0.1 916164 82 88 89 92 <0.1 916184 60 79 87 89 <0.1 916304 46 65 80 84 0.1 916324 57 77 87 92 <0.1 916344 41 70 83 88 0.1 916564 38 66 88 94 0.1 916604 67 87 95 96 <0.1 916624 43 59 79 87 0.1 916803 67 84 93 92 <0.1

TABLE 42 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM) 912759 70 94 98 99 <0.1 915486 35 64 82 90 0.1 915487 62 89 94 95 <0.1 915626 67 83 92 94 <0.1 915786 65 84 88 88 <0.1 916145 53 66 85 87 <0.1 916146 62 77 86 86 <0.1 916165 71 86 89 88 <0.1 916166 71 83 87 88 <0.1 916305 57 86 90 92 <0.1 916306 86 96 98 98 <0.1 916325 59 78 83 86 <0.1 916345 21 47 67 73 0.4 916545 63 88 95 94 <0.1 916546 66 85 92 95 <0.1 916625 47 71 84 92 <0.1 916706 22 65 80 85 0.2 916765 67 85 92 93 <0.1 916845 38 71 80 87 0.1

TABLE 43 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM) 912759 97 99 100 100 <0.1 915608 66 91 97 97 <0.1 915609 71 97 99 99 <0.1 915627 0 26 53 62 1.3 915768 39 69 86 91 0.1 915908 49 70 80 85 <0.1 915987 47 60 75 78 0.1 916008 45 69 84 83 <0.1 916187 71 82 88 92 <0.1 916247 34 72 83 84 0.1 916287 31 70 90 91 0.1 916547 79 93 97 97 <0.1 916566 8 45 73 81 0.5 916586 47 67 89 91 <0.1 916587 48 81 90 94 <0.1 916606 18 64 87 90 0.2 916607 72 94 94 95 <0.1 916627 18 51 82 79 0.3 916805 18 65 78 81 0.2

TABLE 44 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM) 912759 64 92 97 99 <0.1 915610 74 94 98 99 <0.1 915789 35 72 82 85 0.1 915909 52 69 82 86 <0.1 915929 13 32 60 59 1.0 915969 39 54 74 74 0.2 915989 46 67 81 86 0.1 916069 24 59 75 56 0.3 916148 42 71 85 80 <0.1 916168 28 54 74 68 0.3 916188 22 42 72 70 0.4 916309 30 77 91 96 0.1 916348 41 57 65 73 0.1 916549 64 85 94 96 <0.1 916568 54 66 81 87 <0.1 916569 60 86 92 95 <0.1 916728 22 50 68 73 0.4 916788 60 89 94 96 <0.1 916848 49 75 89 95 <0.1

TABLE 45 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM) 912759 81 93 98 100 <0.1 915390 9 40 67 77 0.6 915611 46 80 93 88 <0.1 915630 52 69 81 82 <0.1 915910 44 64 79 80 0.1 915931 83 88 89 86 <0.1 916149 73 87 89 83 <0.1 916150 51 68 77 84 <0.1 916189 60 73 77 79 <0.1 916310 45 77 88 95 <0.1 916330 48 67 84 86 <0.1 916550 62 85 94 97 <0.1 916570 89 96 98 98 <0.1 916629 26 53 73 86 0.3 916630 52 68 87 91 <0.1 916670 43 77 78 85 <0.1 916730 61 74 82 86 <0.1 916768 35 57 67 72 0.2 916789 79 92 96 96 <0.1

TABLE 46 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM) 912759 76 94 96 99 <0.1 915532 31 66 82 92 0.1 915612 54 77 86 90 <0.1 915732 42 63 80 84 0.1 915932 45 71 88 89 <0.1 915951 26 58 71 74 0.3 915991 67 84 85 85 <0.1 915992 54 78 86 87 <0.1 916112 35 67 76 78 0.1 916151 51 79 87 90 <0.1 916311 36 70 81 87 0.1 916331 56 85 93 95 <0.1 916332 82 91 94 96 <0.1 916390 30 41 68 64 0.5 916552 79 93 96 97 <0.1 916571 53 78 90 94 <0.1 916631 48 77 86 90 <0.1 916651 81 89 94 95 <0.1 916711 37 66 85 91 0.1

TABLE 47 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM) 912759 58 90 98 99 <0.1 915474 51 79 90 93 <0.1 915493 48 58 83 80 0.1 915494 46 73 86 90 <0.1 915674 49 72 89 93 <0.1 915933 40 63 75 79 0.1 916153 68 86 89 91 <0.1 916172 85 89 87 87 <0.1 916173 81 90 91 88 <0.1 916292 64 83 92 92 <0.1 916312 60 84 91 92 <0.1 916333 75 92 96 96 <0.1 916572 29 62 79 88 0.2 916592 52 74 89 90 <0.1 916593 25 67 83 93 0.2 916613 46 75 89 92 <0.1 916652 65 83 91 88 <0.1 916672 73 89 93 90 <0.1 916772 50 61 83 89 <0.1

TABLE 48 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM) 912759 50 89 96 99 <0.1 915534 0 33 66 57 1.1 915535 51 81 92 96 <0.1 915634 18 67 79 84 0.2 915635 44 72 86 91 0.1 915675 36 68 82 90 0.1 915735 45 68 73 84 0.1 915936 36 67 78 83 0.1 915995 78 87 90 89 <0.1 915996 83 91 93 92 <0.1 916174 80 84 86 81 <0.1 916175 55 82 86 89 <0.1 916334 50 82 92 94 <0.1 916335 52 76 89 93 <0.1 916575 62 88 93 93 <0.1 916753 49 69 76 74 <0.1 916774 49 72 86 91 <0.1 916794 26 64 85 85 0.2 916873 16 49 72 82 0.4

TABLE 49 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM) 912759 57 90 97 99 <0.1 915477 47 65 88 93 0.1 915478 47 78 90 95 <0.1 915497 63 68 79 86 <0.1 915637 67 91 97 98 <0.1 916037 15 47 70 61 0.6 916236 80 87 90 88 <0.1 916336 52 67 81 87 <0.1 916576 50 76 89 93 <0.1 916596 55 82 93 94 <0.1 916636 42 71 87 90 0.1 916637 56 85 90 93 <0.1 916715 27 38 68 68 0.5 916716 35 77 89 93 0.1 916796 14 62 84 89 0.3 916814 22 44 70 79 0.4 916815 56 79 87 89 <0.1 916816 33 72 83 93 0.1 916874 5 34 61 70 0.8

TABLE 50 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM) 912759 56 91 97 100 <0.1 915479 38 70 89 94 0.1 915618 42 63 75 85 0.1 915619 65 87 96 97 <0.1 915638 31 64 80 82 0.2 915639 33 78 88 93 0.1 915778 41 50 78 87 0.2 916058 26 34 73 81 0.4 916177 38 55 83 82 0.1 916238 84 91 93 93 <0.1 916298 79 87 92 94 <0.1 916318 59 71 91 94 <0.1 916338 71 91 94 92 <0.1 916558 73 89 94 94 <0.1 916577 41 66 78 82 0.1 916578 69 85 91 93 <0.1 916638 46 84 90 92 <0.1 916757 33 60 82 88 0.2 916817 31 67 82 87 0.1

TABLE 51 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM) 841947 50 78 89 92 <0.1 912759 75 50 85 99 <0.1 912986 54 78 90 95 <0.1 915480 61 87 94 97 <0.1 915519 47 77 86 85 <0.1 915620 46 75 88 91 <0.1 915780 24 76 92 94 0.1 915920 23 65 79 82 0.2 916020 45 80 85 81 <0.1 916299 59 87 92 93 <0.1 916339 88 95 97 97 <0.1 916340 83 96 97 98 <0.1 916559 41 68 83 89 0.1 916579 71 86 96 96 <0.1 916580 59 90 95 96 <0.1 916618 10 47 70 78 0.5 916639 38 58 80 86 0.1 916778 68 87 92 94 <0.1 916818 60 77 90 91 <0.1

TABLE 52 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM) 912759 60 0 85 99 0.3 915541 48 71 89 91 <0.1 915542 50 72 86 93 <0.1 915601 8 53 84 84 0.3 915602 1 56 77 91 0.4 915621 21 54 75 80 0.3 915622 0 44 73 84 0.5 915922 27 64 79 85 0.2 916042 6 57 89 88 0.3 916140 43 82 90 89 <0.1 916141 72 88 93 91 <0.1 916180 33 62 69 83 0.2 916181 53 80 89 92 <0.1 916341 0 78 94 94 0.3 916560 72 91 95 94 <0.1 916581 38 76 91 91 0.1 916601 44 80 88 90 <0.1 916701 61 83 91 93 <0.1 916780 75 91 93 94 <0.1

TABLE 53 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 15.6 62.5 250 1,000 IC₅₀ Number nM nM nM nM (μM) 915609 52 86 96 99 <0.01 959430 42 76 90 95 <0.01 959440 39 73 92 98 0.02 959470 46 73 89 94 <0.01 959670 52 90 96 98 <0.01 959680 50 75 91 96 <0.01 959730 83 96 98 98 <0.01 959740 50 70 90 96 <0.01 959820 40 69 85 92 0.02 959830 46 69 93 97 0.02 959880 34 62 85 93 0.03 960010 48 78 92 95 <0.01

TABLE 54 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 15.6 62.5 250 1,000 IC₅₀ Number nM nM nM nM (μM) 915609 53 87 98 99 <0.01 959271 55 74 91 91 <0.01 959360 7 43 79 85 0.1 959361 60 87 93 94 <0.01 959411 56 76 91 94 <0.01 959441 50 81 93 97 <0.01 959460 0 29 75 90 0.2 959701 62 91 97 98 <0.01 959721 80 94 97 97 <0.01 959731 25 64 82 91 0.05 959741 41 65 83 91 0.02 959750 0 26 65 87 0.2 959761 28 60 84 91 0.05 959781 39 58 75 87 0.04 959911 20 54 78 90 0.1 959921 37 61 83 91 0.03 959931 48 72 89 92 <0.01 959960 11 51 79 90 0.1 959961 38 64 85 92 0.03

TABLE 55 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 15.6 62.5 250 1,000 IC₅₀ Number nM nM nM nM (μM) 915609 11 71 93 98 0.1 959412 52 77 90 94 <0.01 959413 34 82 95 97 0.02 959422 15 50 80 87 0.1 959432 33 60 86 95 0.04 959662 0 53 84 92 0.1 959672 54 85 95 97 <0.01 959673 18 62 88 95 0.1 959682 46 77 90 91 <0.01 959702 39 71 91 96 0.02 959703 81 96 99 99 <0.01 959712 4 30 75 92 0.1 959713 0 53 86 96 0.1 959722 33 80 90 94 0.02 959733 31 68 92 96 0.03 959782 35 63 86 94 0.03 959872 29 64 77 89 0.04 959912 25 69 89 92 0.04 959982 21 61 83 91 0.1

TABLE 56 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 15.6 62.5 250 1,000 IC₅₀ Number nM nM nM nM (μM) 915609 2 73 93 98 0.1 959363 49 82 91 91 <0.01 959393 38 71 87 95 0.02 959394 27 73 91 97 0.03 959414 69 94 98 99 <0.01 959664 51 77 95 98 <0.01 959674 43 74 95 98 0.02 959683 14 71 90 96 0.05 959704 57 92 98 99 <0.01 959724 0 68 90 95 0.1 959734 71 93 98 98 <0.01 959814 24 76 90 95 0.03 959873 38 53 83 90 0.04 959874 51 82 95 97 <0.01 959884 44 77 94 97 <0.01 959913 18 50 85 92 0.1 959953 6 51 85 92 0.1 959983 22 54 81 92 0.1 960004 10 71 92 96 0.1

TABLE 57 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 15.6 62.5 250 1,000 IC₅₀ Number nM nM nM nM (μM) 915609 12 69 93 98 0.1 959364 32 72 89 91 0.03 959415 21 70 91 96 0.04 959444 6 47 82 91 0.1 959445 32 70 92 97 0.03 959455 61 87 95 97 <0.01 959675 20 56 80 94 0.1 959684 8 47 83 86 0.1 959705 77 95 98 99 <0.01 959735 12 67 90 95 0.1 959764 4 32 80 92 0.1 959765 1 59 88 93 0.1 959784 3 35 75 90 0.1 959785 27 72 92 96 0.03 959794 0 0 53 83 0.3 959864 26 61 84 91 0.05 959885 49 81 95 96 <0.01 959914 7 43 76 89 0.1 959964 17 55 83 91 0.1

TABLE 58 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 15.6 62.5 250 1,000 IC₅₀ Number nM nM nM nM (μM) 915609 0 73 95 97 0.1 959456 66 90 97 98 <0.01 959666 29 60 89 97 0.04 959676 15 44 81 93 0.1 959686 71 92 97 97 <0.01 959695 40 75 91 93 0.02 959696 21 81 90 92 0.03 959706 81 95 98 98 <0.01 959725 8 55 76 84 0.1 959726 0 59 88 91 0.1 959736 46 84 94 98 <0.01 959766 22 57 83 94 0.1 959776 1 53 87 93 0.1 959815 31 67 89 91 0.03 959865 6 49 84 91 0.1 959875 34 74 91 92 0.02 959935 22 55 84 94 0.1 959955 0 55 83 89 0.1 959985 29 71 88 93 0.03

TABLE 59 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 % Inhibition Compound 15.6 62.5 250 1,000 IC₅₀ Number nM nM nM nM (μm) 915609 37 80 96 99 0.02 959356 40 71 87 88 0.02 959417 25 58 83 92 0.1 959437 65 88 94 95 <0.01 959667 37 69 90 95 0.02 959677 29 56 82 92 0.05 959687 51 79 93 97 <0.01 959697 51 75 93 95 <0.01 959707 81 94 98 98 <0.01 959727 71 92 96 96 <0.01 959737 45 75 89 94 <0.01 959767 47 76 93 96 <0.01 959797 32 59 87 94 0.04 959856 13 35 67 80 0.1 959876 38 75 89 90 0.02 959877 40 81 89 94 <0.01 959956 25 25 66 85 0.1 960006 13 40 68 83 0.1 960007 24 59 88 91 0.05

Example 3: Tolerability of Modified Oligonucleotides Targeting Human PNPLA3 in BALB/c Mice

BALB/c mice are a multipurpose mouse model frequently utilized for safety and efficacy testing. The mice were treated with antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Ionis oligonucleotides selected from the studies above were conjugated with 3′-THA-C₆-GalNAC₃-(3R,5S)-5-(hydroxymethyl) pyrrolidin-3-ol phosphate endcap (henceforth referred to as 3′-THA).

Treatment

Groups of 6- to 7-week-old male mice were injected subcutaneously once with 200 mg/kg of modified oligonucleotides. One group of male BALB/c mice was injected with PBS. Mice were euthanized 72-96 hours after the single dose and plasma was harvested for further analysis.

To evaluate the effect of modified oligonucleotides on liver function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Beckman Coulter AU480, Brea, CA). Modified oligonucleotides that caused changes in the levels of transaminases outside the expected range for antisense oligonucleotides were excluded in further studies. The oligonucleotides which were considered tolerable in this study and were selected for further evaluation are presented in the Table below. ‘Parent Oligo’ indicates the Ionis oligonucleotide that has been described in the studies above and that was conjugated with 3′-THA and tested in this study.

TABLE 60 Antisense oligonucleotides in BALB/c mouse study Compound Parent oligo ID ID 975746 916339 975747 912941 975748 916306 975755 916332 975760 912848 975764 916298 975766 916552 975767 916789 975768 916602 975770 912874 975771 916333 975772 916780 975775 916672 975777 916558 975780 916607 975783 916338 975788 912847 975790 916778 975792 912870 975794 916802 975797 916637 975799 912732 975800 912733 975803 912813 975804 912823 975805 912834 975806 912855 975807 912856 975808 912864 975809 912871 975810 912872 975811 912875 975813 912931 975814 912934 975815 912936 975816 912938 975817 912943 975820 912988 975822 915486 975829 915619 975836 915780 975840 915989 975844 916151 975849 916292 975850 916299 975851 916303 975852 916309 975853 916310 975854 916312 975855 916318 975856 916324 975857 916331 975858 916334 975859 916335 975860 916336 975861 916549 975862 916550 975864 916563 975865 916564 975866 916568 975868 916571 975869 916575 975870 916580 975871 916581 975873 916586 975875 916601 975878 916624 975879 916625 975880 916636 975881 916638 975883 916670 975886 916711 975887 916716 975888 916774 975889 916781 975890 916782 975891 916788 975893 916815 975894 916816 975895 916817 975896 916818 975897 916822 975898 916845 994288 959455 994289 960010 994290 959361 994291 959271

Example 4: Effect of Antisense Inhibition of PNPLA3 in Transgenic Mouse Model

A PNPLA3 transgenic mouse model from wild-type C57BL/6 generated by the University of California, Irvine was used. The mouse model comprises a genomic construct containing the entire PNPLA3 gene fosmid, generously provided by the University of Washington. The efficacy of Ionis oligonucleotides was evaluated in this model.

Treatment

Transgenic mice were maintained on a 12-hour light/dark cycle and were fed ad libitum normal Purina mouse chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

The hPNPLA3 Tg mice were divided into groups of 2 mice each. Groups received subcutaneous injections of Ionis oligonucleotide at a dose of 2.5 mg/kg on days 1 and 8. One group of 4 mice received subcutaneous injections of PBS on days 1 and 8. The saline-injected group served as the control group to which oligonucleotide-treated groups were compared.

RNA Analysis

On day 10, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of PNPLA3. Primer probe sets RTS36070 and RTS36075 were both used to measure PNPLA3 mRNA levels. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN®. As presented in the Table below, treatment with Ionis antisense oligonucleotides resulted in significant reduction of PNPLA3 mRNA in comparison to the PBS control. ‘0’ indicates that the oligonucleotides did not inhibit mRNA expression.

TABLE 61 Percent inhibition of PNPLA3 mRNA in the transgenic mice liver relative to the PBS control Inhibition Inhibition (%) (%) measured measured Compound with with ID RTS36070 RTS36075 975746 99 99 975747 99 99 975748 98 98 975755 99 99 975760 96 97 975764 75 83 975766 99 99 975767 98 98 975768 98 98 975770 97 97 975771 98 99 975772 96 96 975775 90 91 975777 85 89 975780 44 63 975783 87 90 975788 0 26 975790 0 0 975792 9 34 975794 44 50 975797 0 0 975799 0 0 975800 0 5 975803 68 68 975804 11 38 975805 0 0 975806 0 0 975807 0 0 975808 47 58 975809 0 19 975810 12 22 975811 19 32 975813 36 39 975814 48 54 975815 78 77 975816 56 56 975817 84 86 975820 35 45 975822 0 0 975829 98 98 975836 85 91 975840 19 44 975844 21 31 975849 88 89 975850 41 48 975851 5 18 975852 24 41 975853 0 0 975854 0 0 975855 0 0 975856 45 31 975857 73 67 975858 58 40 975860 92 92 975861 66 49 975862 46 36 975864 16 21 975865 0 0 975866 40 41 975868 56 48 975869 30 19 975870 0 14 975871 0 0 975875 75 73 975878 18 12 975879 7 0 975880 0 0 975881 54 54 975883 77 80 975886 18 28 975887 49 57 975888 10 9 975889 90 91 975890 96 98 975891 97 98 975893 95 95 975894 85 87 975895 89 89 975896 91 89 975898 94 95 975897 96 97 975873 99 99 994288 99 99 994289 98 99 994290 98 99 994291 95 95 975859 95 96

Example 5: Tolerability of Modified Oligonucleotides Targeting Human PNPLA3 in CD1 Mice

CD1 ® mice (Charles River, MA) are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were treated with Ionis antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Ionis oligonucleotides selected from the studies above were conjugated with 5′-Trishexylamino-(THA)-C₆GalNAC3 endcap (henceforth referred to as 5′-THA). The Ionis oligonucleotides tested are presented in the Table below. ‘Unconjugated parent ION No.’ refers to the Ionis oligonucleotide described in the in vitro studies above with the same sequence. ‘3′-THA counterpart ION No.’ refers to the 3′-THA conjugated oligonucleotide with the same sequence and evaluated in the mice studies above.

TABLE 62 5′-THA oligonucleotides tested in CD1 mice tolerability study Unconjugated 3′-THA SEQ Compound parent ION counterpart ID ID No. ION No Sequence NO 975591 916339 975746 GGATATATTGGGCTCA 1512 975592 912941 975747 TTGCATTGCATAGCCT  182 975593 916306 975748 GTGTACTTTAGGCTCC  598 975600 916332 975755 CACAATGACATCATGG 1020 975605 912848 975760 CGTTTTTAGTAGTCAA  141 975611 916552 975766 CCTTTTATTTCCGTTA 1024 975612 916789 975767 GTAATATTCAGACCAG  899 975613 916602 975768 CTAGTAAATGCTTGTC  330 975615 912874 975770 ATACTTTTGGCAAGGC  217 975616 916333 975771 CTTTATTCAATGTGGC 1089 975617 916780 975772 AGAAATTGCAGTGCCC 1665 975674 915619 975829 GACTTTAGGGCAGATG 1400 975704 916335 975859 TAATTCTACCTGTGTC 1227 975718 916586 975873 AACTTTGCAGCCTATC  605 975735 916782 975890 CTTAGAAATTGCAGTG  408 975736 916788 975891 TAATATTCAGACCAGG  830 975738 916815 975893 CAATTCTAGACATGGC 1313 975742 916822 975897 TATGACATTTCAGAGT  410 975743 916845 975898 GTAAAGATGTGAGTGA  618 994282 959455 994288 TTGGATATATTGGGCT 1982 994283 960010 994289 AGACATATGACATTTC 1745 994284 959361 994290 TTTTTAGTAGTCAAGG 1757 994285 959271 994291 GTTGAAGGATGGATGG 1748 Treatment

Groups of four CD1 mice each were weekly injected subcutaneously with 15 mg/kg of Ionis oligonucleotides for 6 weeks, with one loading dose at day 4 (total 8 doses). One group of male CD1 mice was injected subcutaneously for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of Ionis oligonucleotides on liver and kidney function, plasma levels of transaminases (ALT and AST), albumin, total bilirubin, and creatinine were measured at week 3 using an automated clinical chemistry analyzer (Beckman Coulter AU480, Brea, CA). The results are presented in the Table below. Ionis oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 63 Plasma chemistry marker levels in CD1 mice at week 3 Total Albumin ALT AST bilirubin Creatinine (g/dL) (IU/L) (IU/L) (mg/dL) (mg/dL) PBS 2.9 31 64 0.4 0.1 975611 2.7 640 385 0.3 0.1 994282 2.4 76 83 0.3 0.1 975592 3.0 786 942 0.5 0.1 975600 2.7 334 431 0.3 0.1 975591 2.6 62 115 0.4 0.1 975718 2.4 1717 2183 1.2 0.1 994284 2.7 41 97 0.3 0.1 994283 2.8 216 154 0.3 0.1 975616 3.0 69 137 0.3 0.1 975612 2.7 47 218 0.4 0.1 975674 2.9 134 114 0.4 0.1 975613 2.8 60 277 0.3 0.1 975593 2.7 429 405 0.4 0.1 975736 2.9 46 63 0.2 0.2 975735 2.5 46 79 0.2 0.1 975742 2.6 152 96 0.2 0.1 975615 2.9 207 189 0.4 0.1 975617 2.9 65 70 0.3 0.1 975605 2.9 67 92 0.3 0.1 975704 2.4 33 61 0.2 0.1 975738 2.6 43 67 0.2 0.1 975743 2.9 119 126 0.4 0.1 994285 2.8 400 353 0.2 0.1 Hematology Assays

Blood obtained from selected mouse groups at week 6 were sent to IDEXX BioResearch for measurement of platelet count. The results are presented in the tables below. Ionis oligonucleotides that caused changes in the platelet count outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 64 Platelet count in CD1 mice Platelet (×10³/μL) PBS 1067 975605 1202 975612 1200 975613 1417 975616 1178 975617 922 975674 618 975591 941 975743 1127 994282 1384 994284 1255 975704 939 975735 1039 975736 1116 975738 1126 975742 808

Example 6: Tolerability of Modified Oligonucleotides Targeting Human PNPLA3 in Sprague-Dawley Rats

Sprague-Dawley rats are a multipurpose model used for safety and efficacy evaluations. The rats were treated with Ionis antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were weekly injected subcutaneously with 15 mg/kg of Ionis oligonucleotide for 6 weeks, with one loading dose on day 4 (total 8 doses). Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of Ionis oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Beckman Coulter AU480, Brea, CA). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in the Table below expressed in IU/L. Plasma levels of bilirubin, creatinine, albumin, and BUN were also measured using the same clinical chemistry analyzer and the results are also presented in the Table below expressed in mg/dL. Ionis oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 65 Plasma chemistry markers in Sprague-Dawley rats Total Albumin ALT AST bilirubin Creatinine BUN (g/dL) (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) PBS 3 35 81 0.2 0.2 12 975591 3 57 161 0.2 0.3 14 975605 4 62 176 0.3 0.2 14 975612 3 106 153 0.2 0.3 13 975613 3 32 94 0.2 0.2 12 975616 4 31 106 0.2 0.3 13 975617 3 49 263 0.2 0.2 12 975735 3 44 128 0.2 0.2 14 975736 3 73 293 0.3 0.3 14 994282 3 41 135 0.1 0.3 12 994284 3 32 95 0.1 0.2 13 Kidney Function

To evaluate the effect of Ionis oligonucleotides on kidney function, urinary levels of protein and creatinine were measured using an automated clinical chemistry analyzer (Beckman Coulter AU480, Brea, CA). The ratios of total protein to creatinine are presented in the Table below. Ionis oligonucleotides that caused changes in the levels of the ratio outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 66 Total protein to creatinine ratio in Sprague-Dawley rats PBS 1.5 975591 2.0 975605 1.6 975612 1.9 975613 2.3 975616 2.0 975617 1.4 975735 2.2 975736 1.1 994282 2.1 994284 2.1 Organ Weights

Liver, heart, spleen and kidney weights were measured at the end of the study, and are presented in the Table below. Ionis oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 67 Organ weights (g) Liver Kidney Spleen Saline 16 3 1 975591 16 4 1 975605 21 3 1 975612 12 3 1 975613 16 3 1 975616 15 3 1 975617 19 4 2 975735 14 4 1 975736 15 3 1 994282 14 3 1 994284 15 3 1

Example 7: Effect of Antisense Inhibition of PNPLA3 in Transgenic Mouse Model

Ionis oligonucleotides were tested in a multi-dose assay in the hPNPLA3 Tg model.

Treatment

Transgenic mice were maintained on a 12-hour light/dark cycle and were fed ad libitum normal Purina mouse chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

Study 1

The hPNPLA3 Tg mice were divided into groups of 4 mice each. Groups received subcutaneous injections of Ionis oligonucleotide at a weekly dose of 5 mg/kg, 1 mg/kg, or 0.25 mg/kg administered on days 1, 5, 8, 15, and 23. One group of 4 mice received subcutaneous injections of PBS on days 1, 5, 8, 15, and 23. The saline-injected group served as the control group to which oligonucleotide-treated groups were compared.

RNA Analysis

On day 26, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of PNPLA3. Primer probe sets RTS36070 and RTS36075 were both used to measure PNPLA3 mRNA levels. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN©. As presented in the Table below, treatment with Ionis antisense oligonucleotides resulted in significant dose-dependent reduction of PNPLA3 mRNA in comparison to the PBS control.

TABLE 68 Percent inhibition of PNPLA3 mRNA in the transgenic mice liver relative to the PBS control Inhibition Inhibition measured measured by by EC₅₀ mg/kg/wk RTS36070 RTS36075 (μg/g) 975605 5 93 90 2.2 1 66 57 0.25 45 46 975612 5 98 99 3.1 1 89 88 0.25 34 44 975613 5 98 97 1.0 1 87 85 0.25 58 56 975616 5 93 93 0.5 1 85 87 0.25 60 63 975617 5 97 97 0.3 1 76 78 0.25 55 53 975735 5 97 98 1.5 1 74 75 0.25 29 33 975736 5 98 98 0.9 1 73 71 0.25 44 45 994282 5 98 98 0.2 1 91 80 0.25 62 58 994284 5 99 100 0.3 1 89 88 0.25 53 47 Study 2

The hPNPLA3 Tg mice were divided into groups of 4 mice each. Groups received subcutaneous injections of Ionis oligonucleotide at a weekly dose of 5 mg/kg, 2.5 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.25 mg/kg administered on days 1, 5, 8, 15, and 23. One group of 4 mice received subcutaneous injections of PBS on days 1, 5, 8, 15, and 23. The saline-injected group served as the control group to which oligonucleotide-treated groups were compared.

RNA Analysis

On day 26, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of PNPLA3. Primer probe sets RTS36070 and RTS36075 were both used to measure PNPLA3 mRNA levels. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN®. As presented in the Table below, treatment with Ionis antisense oligonucleotides resulted in significant dose-dependent reduction of PNPLA3 mRNA in comparison to the PBS control.

TABLE 69 Percent inhibition of PNPLA3 mRNA in the transgenic mice liver relative to the PBS control Inhibition Inhibition measured measured by by EC₅₀ EC₉₀ mg/kg/wk RTS36070 RTS36075 (μg/g) (μg/g) 975612 5 96 97 1.0 8.6 2.5 98 98 1 95 96 0.5 82 83 0.25 43 44 975613 5 99 99 0.9 7.7 2.5 99 99 1 91 91 0.5 82 83 0.25 69 74 975616 5 96 96 1.0 9.4 2.5 94 93 1 89 89 0.5 81 81 0.25 73 60

Example 8: Effect of Modified Oligonucleotides Targeting Human PNPLA3 in Cynomolgus Monkeys

Cynomolgus monkeys were treated with Ionis antisense oligonucleotides selected from studies described in the Examples above. Antisense oligonucleotide tolerability was evaluated.

Treatment

Prior to the study, the monkeys were kept in quarantine during which the animals were observed daily for general health. The monkeys were 2-4 years old and weighed 2-4 kg. Nine groups of 5 randomly assigned male cynomolgus monkeys each were injected subcutaneously with Ionis oligonucleotide or PBS in a clock-wise rotation between four different sites on the back. The monkeys were dosed twice per week (days 1, 5, 9, and 14) for the first 2 weeks, and then subsequently once a week for 10 weeks with 10 mg/kg of Ionis oligonucleotide on days 21, 28, 35, 42, 49, 56, 63, 70, 77, and 84. A control group of 5 cynomolgus monkeys was injected with PBS in a similar manner and served as the control group.

During the study period, the monkeys were observed twice daily for signs of illness or distress. Any animal experiencing more than momentary or slight pain or distress due to the treatment, injury or illness was treated by the veterinary staff with approved analgesics or agents to relieve the pain after consultation with the Study Director. Any animal in poor health or in a possible moribund condition was identified for further monitoring and possible euthanasia. Scheduled euthanasia of the animals was conducted on day 86 approximately 48 hours after the last dose by exsanguination while under deep anesthesia. The protocols described in the Example were approved by the Institutional Animal Care and Use Committee (IACUC).

Body and Organ Weight Measurements

To evaluate the effect of Ionis oligonucleotides on the overall health of the animals, body and organ weights were measured. Body weights and organ weights were measured on day 86 and the data is presented in the Table below. The results indicate that effect of treatment with antisense oligonucleotides on body and organ weights was within the expected range for antisense oligonucleotides. Specifically, treatment with ION 945616 was well tolerated in terms of the body and organ weights of the monkeys.

TABLE 70 Final body and organ weights in cynomolgus monkey Body Liver with Wt Spleen Kidney gallbladder (kg) (g) (g) (g) PBS Control 2797 2.6 13.1 53 994284 2789 3.3 14.7 69 975605 2685 4.1 12.2 58 975616 2868 3.1 12.9 63 994282 2782 4.4 12.1 62 975613 2704 3.0 13.5 60 975617 2761 3.8 14.1 61 975735 2765 4.1 15.5 67 975736 2844 3.0 14.1 66 975612 2711 2.8 13.2 60 Liver Function

To evaluate the effect of Ionis oligonucleotides on hepatic function, blood samples were collected from all the study groups on day 86. The monkeys were fasted overnight prior to blood collection. Blood was collected in tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 minutes and then centrifuged at 3000 rpm for 10 minutes to obtain serum. Levels of various liver function markers were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). Plasma levels of ALT and AST were measured and the results are presented in the Table below, expressed in IU/L. Bilirubin, a liver function marker, was similarly measured and is presented in the Table below, expressed in mg/dL. The results indicate that antisense oligonucleotides had no effect on liver function outside the expected range for antisense oligonucleotides.

TABLE 71 Liver function markers in cynomolgus monkey plasma ALT AST Bilirubin Albumin (IU/L) (IU/L) (mg/dL) (g/dL) PBS Control 38 55 0.2 4.3 994284 64 48 0.2 3.7 975605 48 54 0.3 4.0 975616 54 57 0.3 3.9 994282 89 53 0.3 4.0 975613 60 71 0.4 4.0 975617 65 61 0.3 4.0 975735 59 79 0.3 4.1 975736 70 56 0.3 3.9 975612 61 66 0.3 3.9 Kidney Function

To evaluate the effect of Ionis oligonucleotides on kidney function, blood samples were collected from all the study groups on day 86. The monkeys were fasted overnight prior to blood collection. Blood was collected in tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 minutes and then centrifuged at 3000 rpm for 10 minutes to obtain serum. Levels of BUN and creatinine were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). Results are presented in the Table below, expressed in mg/dL.

The plasma chemistry data indicate that most of the Ionis oligonucleotides did not have any effect on the kidney function outside the expected range for antisense oligonucleotides.

TABLE 72 Plasma BUN and creatinine levels (mg/dL) in cynomolgus monkeys BUN Creatinine PBS Control 23 0.8 994284 24 0.8 975605 27 0.7 975616 21 0.8 994282 24 0.8 975613 23 0.9 975617 21 0.7 975735 20 0.8 975736 23 0.8 975612 20 0.8 Hematology

To evaluate any effect of Ionis oligonucleotides in cynomolgus monkeys on hematologic parameters, blood samples of approximately 0.5 mL of blood was collected from each of the available study animals on day 86. The samples were collected in tubes containing K₂-EDTA. Samples were analyzed for red blood cell (RBC) count, white blood cells (WBC) count, individual white blood cell counts, such as that of monocytes, neutrophils, lymphocytes, as well as for platelet count, hemoglobin content and hematocrit, using an ADVIA2120i hematology analyzer (Siemens, USA).

The data indicate the oligonucleotides did not cause any changes in hematologic parameters outside the expected range for antisense oligonucleotides at this dose.

TABLE 73 Blood cell counts in cynomolgus monkeys RBC Platelets WBC Neutrophils Lymphocytes Monocytes (×10⁶/μL) (×10³/μL) (×10³/μL) (×10³/μL) (×10³/μL) (×10³/μL) PBS Control 6.0 342 12 3.2 7.8 0.3 994284 6.0 410 10 2.7 6.7 0.3 975605 5.8 326 10 4.8 4.5 0.4 975616 6.0 362 10 3.4 5.8 0.3 994282 5.8 359 10 3.9 5.5 0.3 975613 5.5 327 8 2.6 5.5 0.2 975617 6.1 358 10 3.1 6.4 0.3 975735 5.9 241 13 5.4 6.6 0.4 975736 5.8 360 10 3.5 6.4 0.2 975612 6.2 421 11 5.1 5.7 0.2

TABLE 74 Hematologic parameters in cynomolgus monkeys Hemoglobin HCT (g/dL) (%) PBS Control 14 49 994284 14 48 975605 14 46 975616 14 49 994282 14 47 975613 13 46 975617 14 49 975735 14 48 975736 14 48 975612 14 49 Pro-Inflammatory Proteins Analysis

To evaluate any inflammatory effect of Ionis oligonucleotides in cynomolgus monkeys, blood samples were taken for analysis. The monkeys were fasted overnight prior to blood collection. Approximately 1.5 mL of blood was collected from each animal and put into tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 min and then centrifuged at 3,000 rpm for 10 min at room temperature to obtain serum. C-reactive protein (CRP), which is synthesized in the liver and which serves as a marker of inflammation, and complement C₃ were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan).

Example 9: Measurement of Viscosity of Antisense Oligonucleotides Targeting Human PNPLA3

The viscosity of select antisense oligonucleotides from the studies described above was measured with the aim of screening out antisense oligonucleotides which have a viscosity of more than 40 centipoise (cP). Oligonucleotides having a viscosity greater than 40 cP would have less than optimal viscosity.

Oligonucleotides (32-35 mg) were weighed into a glass vial, 120 μL of water was added and the antisense oligonucleotide was dissolved into solution by heating the vial at 50° C. Part (75 μL) of the pre-heated sample was pipetted to a micro-viscometer (Cambridge). The temperature of the micro-viscometer was set to 25° C. and the viscosity of the sample was measured. Another part (20 μL) of the pre-heated sample was pipetted into 10 mL of water for UV reading at 260 nM at 85° C. (Cary UV instrument). The results are presented in the Table below, where the concentration of each antisense oligonucleotide was 200 mg/ml, and indicate that most of the antisense oligonucleotides solutions are optimal in their viscosity under the criterion stated above.

TABLE 75 Viscosity of antisense oligonucleotides at 200 mg/mL Viscosity (cP) 994284 21 975605 19 975616 20 994282 30 975613 24 975617 22 975735 15 975736 49 975612 25

Example 10: Design of Oligonucleotides at the Site of ION 975616

Additional antisense oligonucleotides were designed targeting a PNPLA3 nucleic acid that overlap the target site of ION 916333, which is the unconjugated version of ION 975616, and with different chemical modifications and motifs.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 3-10-3 cEt gapmers or deoxy, MOE, and cEt oligonucleotides. The 3-10-3 cEt gapmers are 16 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three nucleosides. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a cEt sugar modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. The deoxy, MOE and (S)-cEt oligonucleotides are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modification, an (S)-cEt sugar modification, or a deoxy modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; ‘d’ indicates deoxyribose; the number after the ‘d’ indicates the number of deoxyribose; and ‘e’ indicates a MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence (SEQ ID NO: 2).

TABLE 76 Modified oligonucleotides targeting human PNPLA3 Start Stop Compound SEQ Site Site Sequence Number Chemistry ID NO 5599 5614 TCAATGTGGCTTCTAG 995553 kkk-d10-kkk 2170 5600 5615 TTCAATGTGGCTTCTA 959437 kkk-d10-kkk 2089 5601 5616 ATTCAATGTGGCTTCT 959438 kkk-d10-kkk 2171 5602 5617 TATTCAATGTGGCTTC 959439 kkk-d10-kkk 2172 5603 5618 TTATTCAATGTGGCTT 959440 kkk-d10-kkk 1705 5603 5618 TTATTCAATGTGGCTT 995696 k-d10-kekek 1705 5603 5618 TTATTCAATGTGGCTT 995906 kk-d9-eeekk 1705 5603 5618 TTATTCAATGTGGCTT 996116 kk-d9-ekeke 1705 5604 5619 TTTATTCAATGTGGCT 959441 kkk-d10-kkk 1765 5604 5619 TTTATTCAATGTGGCT 995697 k-d10-kekek 1765 5604 5619 TTTATTCAATGTGGCT 995907 kk-d9-eeekk 1765 5604 5619 TTTATTCAATGTGGCT 996117 kk-d9-ekeke 1765 5605 5620 CTTTATTCAATGTGGC 916333 kkk-d10-kkk 1089 5605 5620 CTTTATTCAATGTGGC 995698 k-d10-kekek 1089 5605 5620 CTTTATTCAATGTGGC 995908 kk-d9-eeekk 1089 5605 5620 CTTTATTCAATGTGGC 996118 kk-d9-ekeke 1089 5605 5620 CTTTATTCAATGTGGC 996277 kek-d9-eekk 1089 5606 5621 ACTTTATTCAATGTGG 916334 kkk-d10-kkk 1158 5606 5621 ACTTTATTCAATGTGG 995699 k-d10-kekek 1158 5606 5621 ACTTTATTCAATGTGG 995909 kk-d9-eeekk 1158 5606 5621 ACTTTATTCAATGTGG 996119 kk-d9-ekeke 1158 5607 5622 TACTTTATTCAATGTG 959442 kkk-d10-kkk 1825 5608 5623 TTACTTTATTCAATGT 959443 kkk-d10-kkk 1885

The oligonucleotides were tested in a series of experiments. Cultured A-431 cells at a density of 10,000 cells per well were treated using free uptake with modified oligonucleotides diluted to different concentrations. After a treatment period of approximately 48 hours, PNPLA3 mRNA levels were measured as previously described using the Human PNPLA3 primer-probe set RTS36070. PNPLA3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The IC₅₀ ratios of the assays are presented in the tables below, which is the ratio of the IC₅₀ of a benchmark oligonucleotide to the IC₅₀ of the oligonucleotide. Hence, a bigger value of the ratio indicates that the oligonucleotide is more active than the benchmark.

TABLE XX Efficacy of modified oligonucleotides targeting human PNPLA3 Start Stop Compound IC₅₀ Site Site Number Chemistry ratio 5600 5615 959437 kkk-d10-kkk 1.42 5601 5616 959438 kkk-d10-kkk 0.49 5602 5617 959439 kkk-d10-kkk 0.36 5603 5618 959440 kkk-d10-kkk 0.55 5603 5618 995906 kk-d9-eeekk 1.42 5604 5619 959441 kkk-d10-kkk 1.66 5605 5620 916333 kkk-d10-kkk 1.96 5605 5620 995908 kk-d9-eeekk 0.70 5606 5621 916334 kkk-d10-kkk 0.95 5606 5621 995909 kk-d9-eeekk 1.47 

What is claimed:
 1. A compound comprising a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising the nucleobase sequence SEQ ID NO: 1089, or a pharmaceutically acceptable salt thereof.
 2. The compound of claim 1, wherein the modified oligonucleotide is 16 linked nucleosides having a nucleobase sequence comprising the nucleobase sequence SEQ ID NO: 1089, or a pharmaceutically acceptable salt thereof.
 3. The compound of claim 1, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage, and/or at least one modified sugar, and/or at least one modified nucleobase.
 4. The compound of claim 3, wherein the at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage.
 5. The compound of claim 3, wherein the at least one modified sugar is a bicyclic sugar.
 6. The compound of claim 5, wherein the bicyclic sugar comprises a a 4′-CH₂—O—2′ group or a 4′-CH(CH₃)—O—2′ group or a 4′-(CH₂)₂—O-2′ group.
 7. The compound of claim 3, wherein the at least one modified sugar comprises a 2′-O(CH)₂—OCH₃ group or a 2′-O—CH₃ group.
 8. The compound of claim 3, wherein the at least one modified nucleobase is a 5-methylcytosine.
 9. The compound of claim 1, wherein the modified oligonucleotide comprises: a gap segment consisting of ten linked deoxynucleosides; a 5′ wing segment consisting of three linked nucleosides; and a 3′ wing segment consisting of three linked nucleosides; and wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage; wherein each cytosine is a 5-methylcytosine.
 10. The compound of claim 1, wherein the compound further comprises a conjugate group.
 11. The compound of claim 10, wherein the conjugate group is positioned at the 5′end of the modified oligonucleotide and is


12. The compound of claim 1, wherein the pharmaceutically acceptable salt is a sodium salt.
 13. The compound of claim 1, wherein the pharmaceutically acceptable salt is a potassium salt.
 14. A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier. 